Anti-tnf compounds

ABSTRACT

TNFα antisense oligonucleotides are provided herein. Methods of treating TNFα diseases or disorders using the TNFα antisense oligonucleotides and related products are provided.

RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119 from U.S.provisional application Ser. No. 62/060,424, filed Oct. 6, 2014, thecontents of which are incorporated herein in their entirety.

FIELD OF INVENTION

The invention relates to anti-TNF nucleic acid compounds, as well asmethods of use thereof and compositions thereof.

BACKGROUND OF INVENTION

TNF-α (tumor necrosis factor-alpha) is a pleiotropic cytokine producedby activated macrophages/monocytes and lymphocytes which often promotesinflammatory responses leading to a variety of diseases. TNF-α isreleased from macrophages, monocytes and natural killer cells and playan important role in inflammatory and immune responses, including therecruitment of leukocytes to injured tissues during bacterial and othermicrobial infections, and following stimulation with inflammatorysubstances. When present in excessive quantities, TNF-α is known tocause tissue injury, and has been implicated in the pathology associatedwith inflammatory and autoimmune diseases.

TNF-α mediates biological effects through two distinct membrane-proteinreceptors, TNF-RI and TNF-RII, which differ in sequence and molecularmass. TNF-RI is reported to be present at low levels in most, if notall, human cell types, and expression of the TNF-RI gene in humans canbe upregulated by infection, interferons, and modulators of secondmessengers, such as phorbol esters. The extracellular portions of bothTNF receptors also exist in soluble forms, which are derived frommembrane-bound forms of the receptors by proteolytic cleavage at thecell surface. The soluble TNF receptors retain the ability to bind TNF-αin solution. Soluble TNF receptors have been identified in urine andsera from healthy individuals, and have been shown to be elevated insome chronic diseases and following inoculation with agents that induceTNF-α.

SUMMARY OF INVENTION

In some aspects, the invention is a compound comprising the structuredepicted in FIG. 7 or salts thereof. In some embodiments the compound is18 nucleotides in length. In other embodiments the compound isformulated in a composition with a carrier. In another embodiments, thecompound is a sodium salt. In other embodiments the compound is thestructure depicted in FIG. 8.

In some embodiments the compound further comprises a molecular speciesat one of the ends. In other embodiments the compound further comprisesa molecular species at both ends.

In yet another embodiment the molecular species is selected from thegroup consisting of a spacer, a lipid, a sterol, cholesterol, stearyl,C16 alkyl chain, bile acids, cholic acid, taurocholic acid,deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids,phospholipids, sphingolipids, isoprenoids, such as steroids, vitamins,such as vitamin E, saturated fatty acids, unsaturated fatty acids, fattyacid esters, such as triglycerides, pyrenes, porphyrines, Texaphyrine,adamantane, acridines, biotin, coumarin, fluorescein, rhodamine,Texas-Red, digoxygenin, dimethoxytrityl, t-butyldimethylsilyl,t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy576), Hoechst 33258dye, psoralen, and ibuprofen.

In another embodiment the molecular species is a selected from the groupconsisting of a lipophilic moiety; a folic acid radical; a steroidradical; a carbohydrate radical; a vitamin A radical; a vitamin Eradical; or a vitamin K radical.

In another embodiment the molecular species is connected directly to thecompound through a linkage selected from the group consisting ofphosphodiester, phosphorothioate, methylphosphonate, and amide linkages.In some embodiments the molecular species is connected indirectly to thecompound through a linker.

In other embodiments the linker is a non-nucleotidic linker selectedfrom the group consisting of abasic residues (dSpacer),oligoethyleneglycol, such as triethyleneglycol (spacer 9) orhexaethylenegylcol (spacer 18), and alkane-diol, such as butanediol.

Another aspect of the invention is an oligonucleotide comprisingmUmGmGmGmAmGT*A*G*A*T*G*mAmGmGmUmAmC (SEQ ID NO. 16), wherein theoligonucleotide is 18 nucleotides in length, wherein m is a 2′O methyl,and wherein * is a phosphorothioate modification. In some embodimentsthe oligonucleotide is formulated in a composition with a carrier. Inother embodiments the carrier is a lipid based carrier. In anotherembodiment the carrier is a nanoparticle.

In another embodiment the oligonucleotide further comprises a molecularspecies at the 3′ or 5′ end. In some embodiments the oligonucleotidefurther comprises a molecular species at both the 3′ and 5′ ends.

In other embodiments the molecular species is selected from the groupconsisting of a spacer, a lipid, a sterol, cholesterol, stearyl, C16alkyl chain, bile acids, cholic acid, taurocholic acid, deoxycholate,oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids,sphingolipids, isoprenoids, such as steroids, vitamins, such as vitaminE, saturated fatty acids, unsaturated fatty acids, fatty acid esters,such as triglycerides, pyrenes, porphyrines, Texaphyrine, adamantane,acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red,digoxygenin, dimethoxytrityl, t-butyldimethylsilyl,t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy576), Hoechst 33258dye, psoralen, and ibuprofen.

In some embodiments the molecular species is a selected from the groupconsisting of a lipophilic moiety; a folic acid radical; a steroidradical; a carbohydrate radical; a vitamin A radical; a vitamin Eradical; or a vitamin K radical. In another embodiment the molecularspecies is connected directly to the compound through a linkage selectedfrom the group consisting of phosphodiester, phosphorothioate,methylphosphonate, and amide linkages. In yet another embodiment themolecular species is connected indirectly to the compound through alinker.

In some embodiments the linker is a non-nucleotidic linker selected fromthe group consisting of abasic residues (dSpacer), oligoethyleneglycol,such as triethyleneglycol (spacer 9) or hexaethylenegylcol (spacer 18),and alkane-diol, such as butanediol.

In other aspects the invention is an oligonucleotide comprising 5′TGGGAGTAGATGAGGTAC 3′ (SEQ ID NO. 4), wherein the oligonucleotide is18-19 nucleotides in length, wherein 4-6 nucleotides at the 5′ end and4-6 nucleotides at the 3′ end of the oligonucleotide include a 2′Omethyl, and wherein 4-10 nucleotides have a phosphorothioatemodification. In some embodiments the 6 nucleotides at the 5′ end and 6nucleotides at the 3′ end of the oligonucleotide include a 2′O methyl.In other embodiments 6 nucleotides have a phosphorothioate modification.In another embodiment 7 nucleotides have a phosphorothioatemodification. In yet another embodiment 8 nucleotides have aphosphorothioate modification.

In other embodiments the phosphorothioate modified nucleotides are in acentral region of the oligonucleotide.

In another embodiment the internucleotide linkage associated with theseventh, eighth, ninth, tenth, eleventh, and twelfth nucleotide from the5′ end of the oligonucleotide is phosphorothioate modified.

In another embodiment each nucleotide has either a 2′O methylmodification or phosphorothioate internucleotide linkage. In someembodiments only one nucleotide has both a 2′O methyl modification and aphosphorothioate internucleotide linkage. In other embodiments only onenucleotide has a 2′-modified nucleotide.

In some embodiments the 2′-modification is selected from the group of:2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-0-aminopropyl (2′-O-AP), 2′-0-dimethylaminoethyl (2′-O-DMAOE),2′-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O-N-methylacetamido(2′-O-NMA).

One aspect of the invention is a stable self-assembling nanostructure,comprising an antisense oligonucleotide of 18-19 nucleotides in lengthcomprising TGGGAGTAGATGAGGTAC (SEQ ID NO. 4), wherein a hydrophobicgroup at the 3′ or 5′ terminus self-associates to form the core of thenanostructure in water or other suitable solvents. Self-assemblingnanostructures are generally formed when oligonucleotide is atconcentrations above 5 μM in DNase and RNase free water or othersuitable solvents. In some embodiments the antisense oligonucleotide is18 nucleotides in length. In other embodiments the antisenseoligonucleotide has phosphodiester internucleotide linkages. In anotherembodiment less than all of the internucleotide linkages arephosphodiester.

Another aspect of the invention is a stable self-assemblingnanostructure, comprising an antisense oligonucleotide of 18-19nucleotides in length comprising TGGGAGTAGATGAGGTAC (SEQ ID NO. 4),wherein the antisense oligonucleotide is associated with a core. In someembodiments the antisense oligonucleotide is 18 nucleotides in length.In other embodiments the antisense oligonucleotide has phosphodiesterinternucleotide linkages. In another embodiment less than all of theinternucleotide linkages are phosphodiester.

In other embodiments the antisense oligonucleotide has phosphorothioateinternucleotide linkages. In some embodiments less than all of theinternucleotide linkages are phosphorothioate.

In some embodiments the antisense oligonucleotide has 2′O methylmodifications. In other embodiment less than all of the nucleotidesinclude a 2′O methyl modification.

In another embodiment the antisense oligonucleotide has 17internucleotide linkages and wherein the 6 central internucleotidelinkages are phosphorothioate. In other embodiments the first 6internucleotide linkages at the 5′ end of the oligonucleotide arephosphodiester internucleotide linkages. In yet another embodiment thefirst 6 nucleotides at the 5′ end of the oligonucleotide are 2′O methylmodified nucleotides.

In other embodiments the last 5 nucleotides at the 3′ end of theoligonucleotide are 2′O methyl modified nucleotides.

In another embodiment the antisense oligonucleotide is selected from thegroup consisting of T-G-G-G-A-G-T-A-G-A-T-G-A-G-G-T-A-C-(SEQ ID NO. 4),mUmGmGmGmAmGmUmAmGmAmUmGmAmGmGmUmAmC (SEQ ID NO. 10, Oligo 3742),T*G*G*G*A*G*T*A*G*A*T*G*A*G*G*T*A*C (SEQ ID NO. 9, Oligo 3500),mUmGmGmGmAmGT*A*G*A*T*G*mAmGmGmUmAmC (SEQ ID NO. 16, Oligo 3534), andmU*mG*mG*mG*mA*mG*T*A*G*A*T*G*mA*mG*mG*mU*mA*mC (SEQ ID NO. 18, Oligo3509) wherein—refers to a phosphodiester bond, * refers to aphosphorothioate bond, and m refers to a O methyl.

In another embodiment the antisense oligonucleotide is linked to theexterior of the core. In some embodiments the nanostructure includes2-1,000 copies of the antisense oligonucleotide.

In other embodiments the nanostructure includes at least oneoligonucleotide structurally distinct from the antisenseoligonucleotide.

In some embodiments the antisense oligonucleotide has its 5′-terminusexposed to the outside surface of the nanostructure. In otherembodiments the antisense oligonucleotide has its 3′-terminus exposed tothe outside surface of the nanostructure. In some embodiments theantisense oligonucleotide is positioned laterally on the surface of thenanostructure.

In another embodiment the antisense oligonucleotide is indirectly linkedto the core through a linker. In some embodiments the antisenseoligonucleotide is indirectly linked to the core through more than onelinker.

In some embodiments the core is a solid or hollow core. In otherembodiments the core is inert, paramagnetic or superparamagnetic. Inanother embodiment the core is a solid core.

In other embodiments the solid core is comprised of noble metals,including gold and silver, transition metals including iron and cobalt,metal oxides including silica, polymers or combinations thereof. Inanother embodiments the core is a polymeric core and wherein thepolymeric core is comprised of amphiphilic block copolymers, hydrophobicpolymers including polystyrene, poly(lactic acid), poly(lacticco-glycolic acid), poly(glycolic acid), poly(caprolactone) and otherbiocompatible polymers.

In yet another embodiment the core is a liposomal core.

Another aspect of the invention is a composition comprising thepreviously discussed embodiments of the compound, the oligonucleotide orthe nanostructure, further comprising a therapeutic agent for treating aTNF disorder associated with the nanostructure. In some embodiments thetherapeutic agent is linked to the oligonucleotide.

Each of the compositions and nucleic acids described herein may beformulated in a variety of carriers. In some embodiments the compoundsof the invention are formulated in a topical carrier. In someembodiments the topical formulation is a cream. In other embodiments thetopical formulation is a gel.

Another aspect of the invention is a method for treating a TNF disorder,comprising administering to a subject having a TNF disorder acomposition comprising the previously described aspects of the compound,the oligonucleotide or the nanostructure in an effective amount to treatthe TNF disorder.

In another embodiment the TNF disorder is selected from the groupconsisting of an autoimmune disease, an infectious disease, transplantrejection or graft-versus-host disease, malignancy, a pulmonarydisorder, an intestinal disorder, a cardiac disorder, sepsis, aspondyloarthropathy, a metabolic disorder, anemia, pain, a hepaticdisorder, a skin disorder, a nail disorder, rheumatoid arthritis,psoriasis, psoriasis in combination with psoriatic arthritis, ulcerativecolitis, Crohn's disease, vasculitis, Behcet's disease, ankylosingspondylitis, asthma, chronic obstructive pulmonary disorder (COPD),idiopathic pulmonary fibrosis (IPF), restenosis, diabetes, anemia, pain,a Crohn's disease-related disorder, juvenile rheumatoid arthritis (JRA),a hepatitis C virus infection, psoriatic arthritis, and chronic plaquepsoriasis.

In some embodiments the autoimmune disorder is selected from the groupconsisting of rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis, gouty arthritis, allergy, multiple sclerosis, autoimmunediabetes, autoimmune uveitis, and nephritic syndrome.

In another aspect the invention is a method for reducing TNF levels invivo, comprising administering to a subject a composition comprising thepreviously described embodiments of the compound, the oligonucleotide orthe nanostructure in an effective amount to reduce TNF levels in vivo.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a bar graph showing inhibitory activity in primary humankeratinocytes of several oligonucleotides including Oligo 3482, 3483,3484, 3485, 3486, 3487, and 3488, normalized to Oligo 3495 (a scrambledcontrol) on TNF expression.

FIG. 2 is a bar graph showing inhibitory activity in primary humankeratinocytes of several oligonucleotides including Oligo 3485, 3472,3508, 3500, 3496, 3526, 3607, 3534, 3514, 3509, 3516 and 3495 on TNFexpression.

FIG. 3 is a bar graph showing the effect of various chemistries on theinhibitory activity in primary human keratinocytes of severaloligonucleotides including Oligo 3660, 3743, 3661, 3657, 3669, and 3652compared with their respective controls. The oligonucleotides wereformulated as spherical nucleic acids (SNAs).

FIGS. 4A-4B show the percent gene knockdown of TNF by SNAs in stimulatedhuman keratinocytes. In FIG. 4A, the percent TNF-α mRNA expressionbetween Oligo 3657 and its control, Oligo 3661 is shown graphically.FIG. 4B represents the compounds' half maximal inhibitory concentrations(IC₅₀), 1.8 nM and >100 nM, respectively.

FIGS. 5A-5B show the effects of additional phosphorothioatemodifications on Oligo 3657 modified SNAs. In FIG. 5A, the percentrelative mRNA expression of oligonucleotides with increasingphosphorothioate content is graphed. FIG. 5B shows a table of the halfmaximal effective concentration (EC₅₀) of each oligonucleotide screened.

FIGS. 6A-6B show the effect of hollow SNAs on TNF expression. In FIG.6A, the TNF expression, normalized to untreated is shown, while FIG. 6Bshows the compounds' IC₅₀ values.

FIG. 7 is a structure of a free acid form of a TNF antisenseoligonucleotide.

FIG. 8 is a structure of a salt form of a TNF antisense oligonucleotide.

FIG. 9 shows the percent gene knockdown of TNF by SNAs in stimulatedhuman keratinocytes. The percent TNF mRNA expression between Oligo 6081and its control, Oligo 6093 is shown graphically. Data presented aregene expressions of TNF mRNA relative to cells stimulated with TNF alone(no oligonucleotide, set to 1.0).

FIG. 10 is a bar graph showing the effect of various hexa(ethyleneglycol) spacer lengths on the inhibitory activity of TNF mRNA expressionin stimulated primary human keratinocytes. The percent TNF mRNAexpression of cells treated with Oligos 6080, 6081, 6082 and 6092 andrelative to their respective controls, Oligos 6083, 6093, 6094 and 6095(set to 100) are shown.

FIG. 11 is a bar graph showing L-SNAs knock down TNF in psoriatic exvivo explants.

FIGS. 12A-12D show the reduced clinical and gross pathology scores forS-SNA (self-assembled SNA) and L-SNA (liposomal SNA) forms of TNFantisense oligonucleotides in Inflammatory Bowel Disease (IBD)containing mice. In FIG. 12A, clinical scores of the disease are shownfor the vehicle, TNBS, Oligo 227901 in S-SNA format of various dosingamounts. In FIG. 12B, clinical scores of the disease are shown for thevehicle, TNBS, Oligo 227901 in L-SNA format of various dosing amounts.In FIG. 12C, gross pathology scores of disease are shown for thevehicle, TNBS, Oligo 227901 in S-SNA format of various dosing amounts.In FIG. 12D, gross pathology scores of disease are shown for thevehicle, TNBS, Oligo 227901 in L-SNA format of various dosing amounts. *indicates p<0.05 vs. Vehicle group using One-Way ANOVA followed bypost-hoc Tukey test.

FIG. 13 shows a cryo-TEM of Oligo 6307 self-assembling SNA.

FIGS. 14A-14B depict a schematic representation of cryo-TEM of oligo6307 self-assembling SNA. Schematic of individual (FIG. 14A) andcollection of SNA (FIG. 14B) are shown.

DETAILED DESCRIPTION

The invention in some aspects relates to compositions for reducing TNFαand methods for treating a TNF disorder using those compositions. Highlyeffective TNFα inhibitors have been identified according to aspects ofthe invention. The TNFα inhibitors are nucleic acid based antisensecompositions. The term “TNF-alpha” or “TNF-α” refers to a cytokine thatexists as a 17 kD secreted form and a 26 kD membrane associated form,the biologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules.

A “TNFα inhibitor” as used herein refers to a nucleic acid based agentwhich interferes with TNFα activity. In particular, the TNFα antisenseinhibitors or TNFα antisense oligonucleotides of the invention reducethe expression of the TNFα gene.

The TNF inhibitors of the invention are antisense nucleic acids.Antisense nucleic acids typically include modified or unmodified RNA,DNA, or mixed polymer nucleic acids, and primarily function byspecifically binding to matching sequences resulting in modulation ofpeptide synthesis. Antisense nucleic acids bind to target RNA by WatsonCrick base-pairing and block gene expression by preventing ribosomaltranslation of the bound sequences either by steric blocking or byactivating RNase H enzyme. Antisense molecules may also alter proteinsynthesis by interfering with RNA processing or transport from thenucleus into the cytoplasm.

As used herein, the term “antisense nucleic acid” or “antisenseoligonucleotide” describes a nucleic acid that hybridizes underphysiological conditions to DNA comprising a particular gene or to anmRNA transcript of that gene in this case TNFα and, thereby, inhibitsthe transcription of that gene and/or the translation of that mRNA. Theantisense molecules are designed so as to interfere with transcriptionor translation of a target gene upon hybridization with the target geneor transcript. Those skilled in the art will recognize that the exactlength of the antisense oligonucleotide and its degree ofcomplementarity with its target will depend upon the specific targetselected, including the sequence of the target and the particular baseswhich comprise that sequence. “Inhibition of gene expression” refers tothe absence or observable decrease in the level of protein and/or mRNAproduct from a target gene, such as the TNFα gene. “Specificity” refersto the ability to inhibit the target gene without manifest effects onother genes of the cell. The consequences of inhibition can be confirmedby examination of the outward properties of the cell or organism or bybiochemical techniques such as RNA solution hybridization, nucleaseprotection, Northern hybridization, reverse transcription, geneexpression monitoring with a microarray, antibody binding, enzyme linkedimmunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA),other immunoassays, and fluorescence activated cell analysis (FACS).

The antisense oligonucleotides of the invention inhibit TNFα expression.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% as compared to acell not treated according to the present invention. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell.

In some instances the TNFα inhibitor is a compound having the followingstructure or bioequivalents including salts and prodrugs thereof:

The term bioequivalent compounds, including pharmaceutically acceptablesalts and prodrugs as used herein refers to antisense oligonucleotideshaving the same primary structure as the antisense oligonucleotide ofinterest, but including salt forms or structures which can be cleaved ormodified to have the same type of biological effect as the antisenseoligonucleotide of interest. This is intended to encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof.

“Pharmaceutically acceptable salts” are physiologically andpharmaceutically acceptable salts of the nucleic acids of the invention:i.e., salts that retain the desired biological activity of the compoundof interest and do not impart undesired toxicological effects thereto.Pharmaceutically acceptable salts include but are not limited to (a)salts formed with cations such as sodium, potassium, ammonium,magnesium, calcium, polyamines such as spermine and spermidine, etc.;(b) acid addition salts formed with inorganic acids, for examplehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid and the like; (c) salts formed with organic acids such as,for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid,p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonicacid, and the like; and (d) salts formed from elemental anions such aschlorine, bromine, and iodine.

An example of a salt of the antisense oligonucleotide of the inventionis for example a compound which is a sodium salt having the followingstructure:

The compounds of the invention may also be prepared to be delivered in a“prodrug” form. A “prodrug” is a therapeutic agent that is prepared inan inactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions.

The antisense oligonucleotides of the invention are TNFα antisenseoligonucleotides. An antisense TNFα oligonucleotide refers to a compoundhaving a sequence of nucleotide bases and a subunit-to-subunit backbonethat allows the antisense oligonucleotide to hybridize to a TNFα targetsequence typically by Watson-Crick base pairing, to form an RNA:oligomerheteroduplex within the target sequence.

The specific hybridization of an antisense oligonucleotide with itstarget nucleic acid, TNFα, interferes with the normal function of thenucleic acid, TNFα. This modulation of function of a target nucleic acidby compounds which specifically hybridize to it is generally referred toas “antisense”. The functions of DNA to be interfered with includereplication and transcription. The functions of RNA to be interferedwith include all vital functions such as, for example, translocation ofthe RNA to the site of protein translation, translation of protein fromthe RNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The overall effect of such interference with target nucleic acidfunction is modulation of the expression of TNFα protein. In the contextof the present invention, “modulation” means a decrease or inhibition inthe expression of a gene.

An antisense oligonucleotide “specifically hybridizes” to a targetpolynucleotide if the oligonucleotide hybridizes to the TNFα targetunder physiological conditions, with a thermal melting point (Tm)substantially greater than 37° C., preferably at least 45° C., andtypically 50° C.-80° C. or higher. Such hybridization preferablycorresponds to stringent hybridization conditions, selected to be about10° C., and preferably about 50° C. lower than the Tm for the specificsequence at a defined ionic strength and pH. At a given ionic strengthand pH, the Tm is the temperature at which 50% of a target sequencehybridizes to a complementary polynucleotide.

Polynucleotides are described as “complementary” to one another whenhybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides. A double-stranded polynucleotide can be“complementary” to another polynucleotide, if hybridization can occurbetween one of the strands of the first polynucleotide and the second.Complementarity (the degree that one polynucleotide is complementarywith another) is quantifiable in terms of the proportion of bases inopposing strands that are expected to form hydrogen bonds with eachother, according to generally accepted base-pairing rules. An antisensecompound may be complementary to a target region of a target transcripteven if the two bases sequences are not 100% complementary, as long asthe heteroduplex structure formed between the compound and transcripthas the desired Tm stability.

Identifying an antisense oligonucleotide that targets a particularnucleic acid may be a multistep process. The process usually begins withthe identification of a nucleic acid sequence whose function is to bemodulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular TNFα disorder or disease state. The targeting process alsoincludes determination of a site or sites within this TNFα gene for theantisense interaction to occur such that the desired effect, e.g.,detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the nucleotide sequence 2283-2300 of SEQ IDNO. 34, ie. gtacctca tctactccca (SEQ ID NO. 35).

Preferred antisense oligonucleotides are designed to target human TNFα,for instance, the nucleotide sequence of SEQ ID NO. 34, set forth below.Human TNF-α cDNA sequence has been published by Nedwin, G. E. et al.(Nucleic Acids Res. 1985, 13, 6361-6373); and is disclosed in Genbankaccession number X02910.

(SEQ ID NO. 34)    1gaattccggg tgatttcact cccggctgtc caggcttgtc ctgctacccc acccagcctt   61tcctgaggcc tcaagcctgc caccaagccc ccagctcctt ctccccgcag gacccaaaca  121caggcctcag gactcaacac agcttttccc tccaacccgt tttctctccc tcaacggact  181cagctttctg aagcccctcc cagttctagt tctatctttt tcctgcatcc tgtctggaag  241ttagaaggaa acagaccaca gacctggtcc ccaaaagaaa tggaggcaat aggttttgag  301gggcatgggg acggggttca gcctccaggg tcctacacac aaatcagtca gtggcccaga  361agacccccct cggaatcgga gcagggagga tggggagtgt gaggggtatc cttgatgctt  421gtgtgtcccc aactttccaa atccccgccc ccgcgatgga gaagaaaccg agacagaagg  481tgcagggccc actaccgctt cctccagatg agctcatggg tttctccacc aaggaagttt  541tccgctggtt gaatgattct ttccccgccc tcctctcgcc ccagggacat ataaaggcag  601ttgttggcac acccagccag cagacgctcc ctcagcaagg acagcagagg accagctaag  661agggagagaa gcaactacag accccccctg aaaacaaccc tcagacgcca catcccctga  721caagctgcca ggcaggttct cttcctctca catactgacc cacggcttca ccctctctcc  781cctggaaagg acaccatgag cactgaaagc atgatccggg acgtggagct ggccgaggag  841gcgctcccca agaagacagg ggggccccag ggctccaggc ggtgcttgtt cctcagcctc  901ttctccttcc tgatcgtggc aggcgccacc acgctcttct gcctgctgca ctttggagtg  961atcggccccc agagggaaga ggtgagtgcc tggccagcct tcatccactc tcccacccaa 1021ggggaaatga gagacgcaag agagggagag agatgggatg ggtgaaagat gtgcgctgat 1081agggagggat gagagagaaa aaaacatgga gaaagacggg gatgcagaaa gagatgtggc 1141aagagatggg gaagagagag agagaaagat ggagagacag gatgtctggc acatggaagg 1201tgctcactaa gtgtgtatgg agtgaatgaa tgaatgaatg aatgaacaag cagatatata 1261aataagatat ggagacagat gtggggtgtg agaagagaga tgggggaaga aacaagtgat 1321atgaataaag atggtgagac agaaagagcg ggaaatatga cagctaagga gagagatggg 1381ggagataagg agagaagaag atagggtgtc tggcacacag aagacactca gggaaagagc 1441tgttgaatgc tggaaggtga atacacagat gaatggagag agaaaaccag acacctcagg 1501gctaagagcg caggccagac aggcagccag ctgttcctcc tttaagggtg actccctcga 1561tgttaaccat tctccttctc cccaacagtt ccccagggac ctctctctaa tcagccctct 1621ggcccaggca gtcagtaagt gtctccaaac ctctttccta attctgggtt tgggtttggg 1681ggtagggtta gtaccggtat ggaagcagtg ggggaaattt aaagttttgg tcttggggga 1741ggatggatgg aggtgaaagt aggggggtat tttctaggaa gtttaagggt ctcagctttt 1801tcttttctct ctcctcttca ggatcatctt ctcgaacccc gagtgacaag cctgtagccc 1861atgttgtagg taagagctct gaggatgtgt cttggaactt ggagggctag gatttgggga 1921ttgaagcccg gctgatggta ggcagaactt ggagacaatg tgagaaggac tcgctgagct 1981caagggaagg gtggaggaac agcacaggcc ttagtgggat actcagaacg tcatggccag 2041gtgggatgtg ggatgacaga cagagaggac aggaaccgga tgtggggtgg gcagagctcg 2101agggccagga tgtggagagt gaaccgacat ggccacactg actctcctct ccctctctcc 2161ctccctccag caaaccctca agctgagggg cagctccagt ggctgaaccg ccgggccaat 2221gccctcctgg ccaatggcgt ggagctgaga gataaccagc tggtggtgcc atcagagggc 2281ctgtacctca tctactccca ggtcctcttc aagggccaag gctgcccctc cacccatgtg 2341ctcctcaccc acaccatcag ccgcatcgcc gtctcctacc agaccaaggt caacctcctc 2401tctgccatca agagcccctg ccagagggag accccagagg gggctgaggc caagccctgg 2461tatgagccca tctatctggg aggggtcttc cagctggaga agggtgaccg actcagcgct 2521gagatcaatc ggcccgacta tctcgacttt gccgagtctg ggcaggtcta ctttgggatc 2581attgccctgt gaggaggacg aacatccaac cttcccaaac gcctcccctg ccccaatccc 2641tttattaccc cctccttcag acaccctcaa cctcttctgg ctcaaaaaga gaattggggg 2701cttagggtcg gaacccaagc ttagaacttt aagcaacaag accaccactt cgaaacctgg 2761gattcaggaa tgtgtggcct gcacagtgaa gtgctggcaa ccactaagaa ttcaaactgg 2821ggcctccaga actcactggg gcctacagct ttgatccctg acatctggaa tctggagacc 2881agggagcctt tggttctggc cagaatgctg caggacttga gaagacctca cctagaaatt 2941gacacaagtg gaccttaggc cttcctctct ccagatgttt ccagacttcc ttgagacacg 3001gagcccagcc ctccccatgg agccagctcc ctctatttat gtttgcactt gtgattattt 3061attatttatt tattatttat ttatttacag atgaatgtat ttatttggga gaccggggta 3121tcctggggga cccaatgtag gagctgcctt ggctcagaca tgttttccgt gaaaacggag 3181ctgaacaata ggctgttccc atgtagcccc ctggcctctg tgccttcttt tgattatgtt 3241ttttaaaata tttatctgat taagttgtct aaacaatgct gatttggtga ccaactgtca 3301ctcattgctg agcctctgct ccccagggga gttgtgtctg taatcgccct actattcagt 3361ggcgagaaat aaagtttgct tagaaaagaa acatggtctc cttcttggaa ttaattctgc 3421atctgcctct tcttgtgggt gggaagaagc tccctaagtc ctctctccac aggctttaag 3481atccctcgga cccagtccca tccttagact cctagggccc tggagaccct acataaacaa 3541agcccaacag aatattcccc atcccccagg aaacaagagc ctgaacctaa ttacctctcc 3601ctcagggcat gggaatttcc aactctggga attc

The nanostructures descried herein may be stable self-assemblingnanostructures. For instance the nanostructure may be an antisenseoligonucleotide of 18-19 nucleotides in length comprisingTGGGAGTAGATGAGGTAC (SEQ ID NO. 4), wherein a hydrophobic group at the 3′or 5′ terminus self-associates to form the core of the nanostructure inwater or other suitable solvents. A hydrophobic group as used herein mayinclude cholesterol, a cholesteryl or modified cholesteryl residue,adamantine, dihydrotesterone, long chain alkyl, long chain alkenyl, longchain alkynyl, olely-lithocholic, cholenic, oleoyl-cholenic, palmityl,heptadecyl, myrisityl, bile acids, cholic acid or taurocholic acid,deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids,phospholipids, sphingolipids, isoprenoids, such as steroids, vitamins,such as vitamin E, fatty acids either saturated or unsaturated, fattyacid esters, such as triglycerides, pyrenes, porphyrines, Texaphyrine,adamantane, acridines, biotin, coumarin, fluorescein, rhodamine,Texas-Red, digoxygenin, dimethoxytrityl, t-butyldimethylsilyl,t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy5), Hoechst 33258 dye,psoralen, or ibuprofen.

The antisense oligonucleotides typically have a length of 15-20 bases,which is generally long enough to have one complementary sequence in themammalian genome. Additionally, antisense compounds having a length ofat least 12, typically at least 15 nucleotides in length hybridize wellwith their target mRNA. Thus, the antisense oligonucleotides of theinvention are typically in a size range of 8-100 nucleotides, morepreferably 12-50 nucleotides in length. In some embodiments of theinvention the antisense oligonucleotides are of 18-19 nucleotides inlength and comprise TGGGAGTAGATGAGGTAC (SEQ ID NO. 4). Antisenseoligonucleotides that comprise SEQ ID NO. 4 may include furthernucleotides on the 5′ and/or 3′ end of the oligonucleotide. However anantisense oligonucleotide that comprises SEQ ID NO. 4 and is limited to18 nucleotides in length does not have any additional nucleotides on the5′ or 3′ end of the molecule. Other non-nucleotide molecules may belinked covalently or non-covalently to the 5′ and/or 3′ end of the thoseoligonucleotides.

In some instances, the antisense oligonucleotide is one of the followingoligonucleotides: T-G-G-G-A-G-T-A-G-A-T-G-A-G-G-T-A-C (SEQ ID NO. 4),mUmGmGmGmAmGmUmAmGmAmUmGmAmGmGmUmAmC (SEQ ID NO. 10, Oligo 3742),T*G*G*G*A*G*T*A*G*A*T*G*A*G*G*T*A*C (SEQ ID NO. 9, Oligo 3500),mUmGmGmGmAmGT*A*G*A*T*G*mAmGmGmUmAmC (SEQ ID NO. 16, Oligo 3534), andmU*mG*mG*mG*mA*mG*T*A*G*A*T*G*mA*mG*mG*mU*mA*mC (SEQ ID NO. 18, Oligo3509) wherein—refers to a phosphodiester bond, * refers to aphosphorothioate bond, and m refers to a O methyl.

The terms “nucleic acid” and “oligonucleotide” are used interchangeablyto mean multiple nucleotides (i.e., molecules comprising a sugar (e.g.,ribose or deoxyribose) linked to a phosphate group and to anexchangeable organic base, which is either a substituted pyrimidine(e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine(e.g., adenine (A) or guanine (G)). As used herein, the terms “nucleicacid” and “oligonucleotide” refer to oligoribonucleotides as well asoligodeoxyribonucleotides. The terms “nucleic acid” and“oligonucleotide” shall also include polynucleosides (i.e., apolynucleotide minus the phosphate) and any other organic basecontaining polymer. Nucleic acid molecules are preferably synthetic(e.g., produced by nucleic acid synthesis). The oligonucleotides may beany size useful for producing antisense effects. In some embodimentsthey are 18-23 nucleotides in length. In other embodiments the antisenseoligonucleotide is 18 nucleotides in length.

The terms “nucleic acid” and “oligonucleotide” may also encompassnucleic acids or oligonucleotides with substitutions or modifications,such as in the bases and/or sugars. For example, they include nucleicacids having backbone sugars that are covalently attached to lowmolecular weight organic groups other than a hydroxyl group at the 2′position and other than a phosphate group or hydroxy group at the 5′position. Thus modified nucleic acids may include a 2′-O-alkylatedribose group. In addition, modified nucleic acids may include sugarssuch as arabinose or 2′-fluoroarabinose instead of ribose. Thus thenucleic acids may be heterogeneous in backbone composition therebycontaining any possible combination of polymer units linked togethersuch as peptide-nucleic acids (which have an amino acid backbone withnucleic acid bases). Other examples are described in more detail below.

The oligonucleotides may be DNA, RNA, PNA, LNA, ENA or hybrids includingany chemical or natural modification thereof. Chemical and naturalmodifications are well known in the art. Such modifications include, forexample, modifications designed to increase binding to a target strand(i.e., increase their melting temperatures), to assist in identificationof the oligonucleotide or an oligonucleotide-target complex, to increasecell penetration, to stabilize against nucleases and other enzymes thatdegrade or interfere with the structure or activity of theoligonucleotides, to provide a mode of disruption (a terminating event)once sequence-specifically bound to a target, and to improve thepharmacokinetic properties of the oligonucleotide.

Modifications include, but are not limited to, for example, (a) endmodifications, e.g., 5′ end modifications (phosphorylationdephosphorylation, conjugation, inverted linkages, etc.), 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with modified bases,stabilizing bases, destabilizing bases, or bases that base pair with anexpanded repertoire of partners, or conjugated bases, (c) sugarmodifications (e.g., at the 2′ position or 4′ position) or replacementof the sugar, as well as (d) internucleoside linkage modifications,including modification or replacement of the phosphodiester linkages. Tothe extent that such modifications interfere with translation (i.e.,results in a reduction of 50%, 60%, 70%, 80%, or 90% or more intranslation relative to the lack of the modification—e.g., in an invitro translation assay), the modification may not be optimal for themethods and compositions described herein.

Non-limiting examples of modified internucleoside linkages includephosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Modified internucleoside linkages that do not include a phosphorus atomtherein have internucleoside linkages that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatoms andalkyl or cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Substituted sugar moieties include, but are not limited to one of thefollowing at the 2′ position: H (deoxyribose); OH (ribose); F; 0-, S-,or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl.

A chemically or naturally modified oligonucleotide may include, forexample, at least one nucleotide modified at the 2′ position of thesugar, most preferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or2′-fluoro-modified nucleotide or an end cap. In other embodiments, RNAmodifications include 2′-fluoro, 2′-amino and 2′ O-methyl modificationson the ribose of pyrimidines, abasic residues or an inverted base at the3′ end of the RNA.

The oligonucleotides useful according to the invention may include asingle modified nucleoside. In other embodiments the oligonucleotide mayinclude at least two modified nucleosides, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 15, at least 20 or more nucleosides, up to the entire length ofthe oligonucleotide.

Nucleosides or nucleobases include the natural purine bases adenine (A)and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleosides include other synthetic and naturalnucleobases such as inosine, xanthine, hypoxanthine, nubularine,isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine,2-(propyl)adenine, 2 (amino)adenine, 2-(aminoalkyll)adenine, 2(aminopropyl)adenine, 2 (methylthio) N6 (isopentenyl)adenine, 6(alkyl)adenine, 6 (methyl)adenine, 7 (deaza)adenine, 8 (alkenyl)adenine,8-(alkyl)adenine, 8 (alkynyl)adenine, 8 (amino)adenine, 8-(halo)adenine,8-(hydroxyl)adenine, 8 (thioalkyl) adenine, 8-(thiol)adenine,N6-(isopentyl)adenine, N6 (methyl)adenine, N6, N6 (dimethyl)adenine,2-(alkyl)guanine,2 (propyl)guanine, 6-alkyl)guanine, 6 (methyl)guanine,7 (alkyl)guanine, 7 (methyl)guanine, 7 (deaza)guanine, 8 (alkyl)guanine,8-(alkenyl)guanine, 8 (alkynyl)guanine, 8-(amino)guanine, 8(halo)guanine, 8-(hydroxyl)guanine, 8 (thioalkyl)guanine,8-(thiol)guanine, N (methyl)guanine, 2-(thio)cytosine, 3 (deaza) 5(aza)cytosine, 3-(alkyl)cytosine, 3 (methyl)cytosine, 5-(alkyl)cytosine,5-(alkynyl)cytosine, 5 (halo)cytosine, 5 (methyl)cytosine, 5(propynyl)cytosine, 5 (propynyl)cytosine, 5 (trifluoromethyl)cytosine,6-(azo)cytosine, N4 (acetyl)cytosine, 3 (3 amino-3 carboxypropyl)uracil,2-(thio)uracil, 5 (methyl) 2 (thio)uracil, 5 (methylaminomethyl)-2(thio)uracil, 4-(thio)uracil, 5 (methyl) 4 (thio)uracil, 5(methylaminomethyl)-4 (thio)uracil, 5 (methyl) 2,4 (dithio)uracil, 5(methylaminomethyl)-2,4 (dithio)uracil, 5 (2-aminopropyl)uracil,5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5(aminoallyl)uracil, 5 (aminoalkyl)uracil, 5 (guanidiniumalkyl)uracil, 5(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil,5-(dialkylaminoalkyl)uracil, 5 (dimethylaminoalkyl)uracil,5-(halo)uracil, 5-(methoxy)uracil, uracil-5 oxyacetic acid, 5(methoxycarbonylmethyl)-2-(thio)uracil, 5(methoxycarbonyl-methyl)uracil, 5 (propynyl)uracil, 5 (propynyl)uracil,5 (trifluoromethyl)uracil, 6 (azo)uracil, dihydrouracil, N3(methyl)uracil, 5-uracil (i.e., pseudouracil), 2 (thio)pseudouracil, 4(thio)pseudouraci1,2,4-(dithio)psuedouracil, 5-(alkyl)pseudouracil,5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil,5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4 (thio)pseudouracil,5-(methyl)-4 (thio)pseudouracil, 5-(alkyl)-2,4 (dithio)pseudouracil,5-(methyl)-2,4 (dithio)pseudouracil, 1 substituted pseudouracil, 1substituted 2(thio)-pseudouracil, 1 substituted 4 (thio)pseudouracil, 1substituted 2,4-(dithio)pseudouracil, 1(aminocarbonylethylenyl)-pseudouracil, 1(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1(aminocarbonylethylenyl)-4 (thio)pseudouracil, 1aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1(arninoalkylaminocarbonylethylenyl)-pseudouracil, 1(arninoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil,1(arninoalkylarninocarbonylethylenyl)-4 (thio)pseudouracil, 1(arninoalkylarninocarbonylethylenyl)-2,4-(dithio)pseudouracil,1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,7-(arninoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine,nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl,7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl,nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl,3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl,3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl,6-(methyl)-7-(aza)indolyl, imidizopyridinyl,9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl,2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl,phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl,tetracenyl, pentacenyl, diiluorotolyl,4-(iluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole,6-(azo)thymine, 2-pyridinone, 5 nitroindole, 3 nitropyrrole,6-(aza)pyrimidine, 2 (amino)purine, 2,6-(diamino) purine, 5 substitutedpyrimidines, N2-substituted purines, N6-substituted purines,06-substituted purines, substituted 1,2,4-triazoles,pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl,2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylatedderivatives thereof.

The antisense oligonucleotides of the invention may be chimericoligonucleotides. Chimeric antisense compounds of the invention may beformed as composite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleotides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. In particular a gapmer is an oligonucleotide thathas at least three discrete portions, two of which are similar i.e.include one or more backbone modifications, and surround a region thatis distinct, i.e., does not include backbone modifications.

The oligonucleotides may include a molecular species at one or bothends, i.e., at the 3′ and/or 5′ end. A molecular species as used hereinrefers to any compound that is not a naturally occurring ornon-naturally occurring nucleotide. Molecular species include but arenot limited to a spacer, a lipid, a sterol, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, an octadecylamine or hexylamino-carbonyl-oxycholesterolmoiety, stearyl, C16 alkyl chain, bile acids, cholic acid, taurocholicacid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid,glycolipids, phospholipids, sphingolipids, isoprenoids, such assteroids, vitamins, such as vitamin E, saturated fatty acids,unsaturated fatty acids, fatty acid esters, such as triglycerides,pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin,coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin,dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyaninedyes (e.g. Cy3 or Cy576), Hoechst 33258 dye, psoralen, or ibuprofen.

The molecular species may be attached at various positions of theoligonucleotide. As described above, the molecular species may be linkedto the 3′-end or 5′-end of the oligonucleotide, where it also serves thepurpose to enhance the stability of the oligomer against 3′- or5′-exonucleases. Alternatively, it may be linked to an internalnucleotide or a nucleotide on a branch. The molecular species may beattached to a 2′-position of the nucleotide. The molecular species mayalso be linked to the heterocyclic base of the nucleotide.

The molecular species may be connected to the oligonucleotide by alinker moiety. Optionally the linker moiety is a non-nucleotidic linkermoiety. Non-nucleotidic linkers are e.g. abasic residues (dSpacer),oligoethyleneglycol, such as triethyleneglycol or hexaethylenegylcol, oralkane-diol, such as butanediol. The spacer units are preferably linkedby phosphodiester or phosphorothioate bonds. The linker units may appearjust once in the molecule or may be incorporated several times, e.g. viaphosphodiester, phosphorothioate, methylphosphonate, or amide linkages.

The oligonucleotide of the invention (separate from the linkersconnecting nucleotides to the molecular species) may also containnon-nucleotidic linkers, in particular abasic linkers (dSpacers),trietyhlene glycol units or hexaethylene glycol units. Further preferredlinkers are alkylamino linkers, such as C3, C6, C12 aminolinkers, andalso alkylthiol linkers, such as C3 or C6 thiol linkers.

TNFα plays a role in a wide variety of TNFα-related disorders. A TNFαdisorder as used herein refers to a disorder in which TNFα activity isdetrimental to a particular physiological function in a subject. As usedherein, the term “a disorder in which TNFα activity is detrimental” isintended to include diseases and other disorders in which the levels ofTNFα expressed in a subject suffering from the disorder plays a role inthe pathophysiology of the disorder or as a factor that contributes to aworsening of or maintenance of the disorder. Accordingly, a disorder inwhich TNFα activity is detrimental is a disorder in which inhibition ofTNFα activity is expected to alleviate at least one symptom and/orprogression or worsening of the disorder. Such disorders may beevidenced, for example, by an increase in the concentration of TNFα in abiological fluid of a subject suffering from the disorder (e.g., anincrease in the concentration of TNFα in serum, plasma, synovial fluid,etc. of the subject), which can be detected, for example, using a TNFαprobe or an anti-TNFα antibody for detecting TNFα message or proteinrespectively.

TNFα disorders include but are not limited to sepsis, infections,autoimmune diseases, cancer, transplant rejection and graft-versus-hostdisease, transplant rejection, malignancy, a pulmonary disorder, anintestinal disorder, a cardiac disorder, sepsis, a spondyloarthropathy,a metabolic disorder, anemia, pain, a hepatic disorder, a skin disorder,a nail disorder, rheumatoid arthritis, psoriasis, psoriasis incombination with psoriatic arthritis, ulcerative colitis, Crohn'sdisease, vasculitis, Behcet's disease, ankylosing spondylitis, asthma,chronic obstructive pulmonary disorder (COPD), idiopathic pulmonaryfibrosis (IPF), restenosis, diabetes, anemia, pain, a Crohn'sdisease-related disorder, juvenile rheumatoid arthritis (JRA), ahepatitis C virus infection, psoriatic arthritis, and chronic plaquepsoriasis.

The biological role played by TNFα in several of these diseases isdescribed below. Inhibiting TNFα expression in these diseases provides atherapeutic treatment for the disorder. TNFα plays a role in sepsis.Biological effects include hypotension, myocardial suppression, vascularleakage syndrome, organ necrosis, stimulation of the release of toxicsecondary mediators and activation of the clotting cascade.

TNFα has been implicated in autoimmune disease, for example, byactivating tissue inflammation and causing joint destruction inrheumatoid arthritis, promoting the death of islet cells and inmediating insulin resistance in diabetes, mediating cytotoxicity tooligodendrocytes and induction of inflammatory plaques in multiplesclerosis, mediating cytotoxicity to oligodendrocytes and induction ofinflammatory plaques in multiple sclerosis, and in the development ofCrohn's disease.

The biological effects observed in a variety of infectious diseases aredue to TNFα. For example, TNFα has been implicated in mediating braininflammation and capillary thrombosis and infarction in malaria,mediating brain inflammation, inducing breakdown of the blood-brainbarrier, triggering septic shock syndrome and activating venousinfarction in meningitis, and in inducing cachexia, stimulating viralproliferation and mediating central nervous system injury in acquiredimmune deficiency syndrome (AIDS).

TNFα has also been implicated as a key mediator of allograft rejectionand graft versus host disease (GVHD) and in mediating an adversereaction that has been observed when the rat antibody OKT3, directedagainst the T cell receptor CD3 complex, is used to inhibit rejection ofrenal transplants.

TNFα has been implicated in inducing cachexia, stimulating tumor growth,enhancing metastatic potential and mediating cytotoxicity inmalignancies.

Pulmonary disorders are also linked to TNFα. For instance, TNFα plays arole in adult respiratory distress syndrome (ARDS), includingstimulating leukocyte-endothelial activation, directing cytotoxicity topneumocytes and inducing vascular leakage syndrome. The compositions ofthe invention can be used to treat various pulmonary disorders,including adult respiratory distress syndrome, shock lung, chronicpulmonary inflammatory disease, pulmonary sarcoidosis, pulmonaryfibrosis, silicosis, idiopathic interstitial lung disease and chronicobstructive airway disorders. Examples of chronic obstructive airwaydisorders include asthma and Chronic Obstructive Pulmonary Disease(COPD).

Inflammatory bowel disorders including Crohn's disease are alsoassociated with TNF. Examples of Crohn's disease-related disordersinclude fistulas in the bladder, vagina, and skin; bowel obstructions;abscesses; nutritional deficiencies; complications from corticosteroiduse; inflammation of the joints; erythem nodosum; pyoderma gangrenosum;and lesions of the eye.

TNFα plays a role in cardiac or coronary disorders, including ischemiaof the heart. A cardiac disorder in which TNFα activity is detrimentalis intended to include coronary and cardiovascular diseases in which thepresence of TNFα in a subject suffering from the disorder has been shownto be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder, including cardiovascular disorders, e.g.,restenosis. These disorders refer to any disease, disorder, or stateinvolving the cardiovascular system, e.g., the heart, the blood vessels,and/or the blood. Examples of a cardiovascular disorder include, but arenot limited to, coronary artery disease, angina pectoris, myocardialinfarction, cardiovascular tissue damage caused by cardiac arrest,cardiovascular tissue damage caused by cardiac bypass, cardiogenicshock, and hypertension, atherosclerosis, coronary artery spasm,coronary artery disease, valvular disease, arrhythmias, andcardiomyopathies.

Inflammatory diseases such as spondyloarthopathies are also aggravatedby TNFα. Spondyloarthropathy refers to any one of several diseasesaffecting the joints of the spine, wherein such diseases share commonclinical, radiological, and histological features.

Metabolic disorders, such as diabetes and obesity have been linked toTNFα. The term metabolic disorder refers to diseases or disorders whichaffect how the body processes substances needed to carry outphysiological functions. Examples of diabetes include type 1 diabetesmellitus, type 2 diabetes mellitus, diabetic neuropathy, peripheralneuropathy, diabetic retinopathy, diabetic ulcerations, retinopathyulcerations, diabetic macrovasculopathy, and obesity.

TNFα has been implicated in the development of anemias. An anemia is anabnormally low number of circulating red cells or a decreasedconcentration of hemoglobin in the blood. Examples of anemia related torheumatoid arthritis include, for example, anemia of chronic disease,iron deficiency anemia, and autoimmune hemolytic anemia.

TNFα has also been implicated in a wide variety of pain syndromes. Theterm “pain” as used herein, refers to all types of pain including acuteand chronic pains, such as neuropathic pain and post-operative pain,chronic lower back pain, cluster headaches, herpes neuralgia, phantomlimb pain, central pain, dental pain, opioid-resistant pain, visceralpain, surgical pain, bone injury pain, pain during labor and delivery,pain resulting from burns, including sunburn, post-partum pain,migraine, angina pain, and genitourinary tract-related pain includingcystitis. The term also includes nociceptive pain or nociception.

Hepatic disorders are also associated with TNFα. Hepatic disordersinclude diseases and other disorders of the liver or conditionsassociated with hepatocellular injury or a biliary tract disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Hepatic disorders include disordersassociated with hepatocellular injuries, including alcoholic cirrhosis,ca antitrypsin deficiency, autoimmune cirrhosis, cryptogenic cirrhosis,fulminant hepatitis, hepatitis B and C, and steatohepatitis. Examples ofbiliary tract disorders include cystic fibrosis, primary biliarycirrhosis, sclerosing cholangitis and biliary obstruction

TNFα has been implicated in skin and nail disorders. A skin disorderrefers to abnormalities, other than injury wounds, of the skin involvinginflammation. Examples of skin disorders include, but are not limitedto, psoriasis, pemphigus vulgaris, scleroderma, atopic dermatitis,sarcoidosis, erythema nodosum, hidradenitis suppurative, lichen planus,Sweet's syndrome, and vitiligo.

TNFα has been implicated in vasculitides, a group of disorders which arecharacterized by the inflammation of blood vessels. Examples ofvasculitides in which TNFα activity is detrimental, include but are notlimited to Behcet's disease, large vessel diseases such as giant cellarteritis, polymyalgia rheumatica, and Takayasu's disease or arteritis,medium vessel diseases such as classic polyarteritis nodosa andKawasaki's disease or small vessel diseases such as Behcet's Syndrome,Wegner's granulomatosis, microscopic polyangitis, hypersensitivityvasculitis, small vessel vasculitis, Henoch-Schonlein purpura, allergicgranulamotosis and vasculitis, and isolated central nervous systemvasculitis, and thromboangitis obliterans.

Various other disorders in which TNFα activity is detrimental includebut are not limited to juvenile arthritis, endometriosis, prostatitis,choroidal neovascularization, sciatica, Sjogren's Syndrome, uveitis, wetmacular degeneration, osteoporosis, osteoarthritis, inflammatory bonedisorders, bone resorption disease, coagulation disturbances, burns,reperfusion injury, keloid formation, scar tissue formation, pyrexia,periodontal disease, obesity, radiation toxicity, age-related cachexia,Alzheimer's disease, brain edema, inflammatory brain injury, cancer,chronic fatigue syndrome, dermatomyositis, drug reactions, such asStevens-Johnson syndrome and Jarisch-Herxheimer reaction, edema inand/or around the spinal cord, familial periodic fevers, Felty'ssyndrome, fibrosis, glomerulonephritides (e.g. post-streptococcalglomerulonephritis or IgA nephropathy), loosening of prostheses,microscopic polyangiitis, mixed connective tissue disorder, multiplemyeloma, cancer and cachexia, multiple organ disorder, myelo dysplasticsyndrome, orchitism osteolysis, pancreatitis, including acute, chronic,and pancreatic abscess, polymyositis, progressive renal failure,pseudogout, pyoderma gangrenosum, relapsing polychondritis, rheumaticheart disease, sarcoidosis, sclerosing cholangitis, stroke,thoracoabdominal aortic aneurysm repair (TAAA), TNF receptor associatedperiodic syndrome (TRAPS), symptoms related to Yellow Fever vaccination,inflammatory diseases associated with the ear, chronic ear inflammation,chronic otitis media with or without cholesteatoma, pediatric earinflammation, myotosis, ovarian cancer, colorectal cancer, therapyassociated with induced inflammatory syndrome (e.g., syndromes followingIL-2 administration), and disorders associated with a reperfusioninjury.

The oligonucleotides may be administered alone or in conjunction withanother therapeutic agent for the treatment of a TNFα disorder.Nonlimiting examples of therapeutic agents with which the TNFα inhibitorof the invention can be combined include the following: non-steroidalanti-inflammatory drug(s) (NSAIDs); cytokine suppressiveanti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanizedanti-TNFα antibody; Celltech/Bayer); cA2/infliximab (chimeric anti-TNFαantibody; Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNF receptor-IgGfusion protein; Immunex; 55 kdTNF-IgG (55 kD TNF receptor-IgG fusionprotein; Hoffmann-LaRoche); IDEC-CE9/SB 210396 (non-depleting primatizedanti-CD4 antibody; IDEC/SmithKline; DAB 486-IL-2 and/or DAB 389-IL-2(IL-2 fusion proteins; Seragen; Anti-Tac (humanized anti-IL-2Ra; ProteinDesign Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering);IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine;DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists (e.g., agonistantibodies); IL-1 RA (IL-1 receptor antagonist; Synergen/Amgen);anakinra (Kineret/Amgen); TNF-bp/s-TNF (soluble TNF binding protein);R973401 (phosphodiesterase Type IV inhibitor; MK-966 (COX-2 Inhibitor;Iloprost; methotrexate; thalidomide and thalidomide-related drugs (e.g.,Celgen); leflunomide (anti-inflammatory and cytokine inhibitor;tranexamic acid (inhibitor of plasminogen activation; T-614 (cytokineinhibitor; prostaglandin E1; Tenidap (non-steroidal anti-inflammatorydrug; Naproxen (non-steroidal anti-inflammatory drug; Meloxicam(non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidalanti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatorydrug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin(non-steroidal anti-inflammatory drug); Sulfasalazine; Azathioprine; ICEinhibitor (inhibitor of the enzyme interleukin-1-beta-convertingenzyme); zap-70 and/or Ick inhibitor (inhibitor of the tyrosine kinasezap-70 or Ick); VEGF inhibitor and/or VEGF-R inhibitor (inhibitors ofvascular endothelial cell growth factor or vascular endothelial cellgrowth factor receptor; inhibitors of angiogenesis); corticosteroidanti-inflammatory drugs (e.g., SB203580); TNF-convertase inhibitors;anti-IL-12 antibodies; anti-IL-18 antibodies; interleukin-11;interleukin-13; interleukin-17 inhibitors; gold; penicillamine;chloroquine; hydroxychloroquine; chlorambucil; cyclosporine;cyclophosphamide; total lymphoid irradiation; anti-thymocyte globulin;anti-CD4 antibodies; CD5-toxins; orally-administered peptides andcollagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs) HP228and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisensephosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals,Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.);prednisone; orgotein; glycosaminoglycan polysulphate; minocycline;anti-IL2R antibodies; marine and botanical lipids (fish and plant seedfatty acids; auranofin; phenylbutazone; meclofenamic acid; flufenamicacid; intravenous immune globulin; zileuton; azaribine; mycophenolicacid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose(therafectin); cladribine (2-chlorodeoxyadenosine); methotrexate;antivirals; and immune modulating agents.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the present invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography. A number of studies have examined the optimal dosagesfor antisense oligonucleotides.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein.

Subject doses of the compounds described herein typically range fromabout 0.1 μg to 10,000 mg, more typically from about 1μg/day to 8000 mg,and most typically from about 10 μg to 100 μg. Stated in terms ofsubject body weight, typical dosages range from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above. The absolute amount will depend upon a varietyof factors including the concurrent treatment, the number of doses andthe individual patient parameters including age, physical condition,size and weight. These are factors well known to those of ordinary skillin the art and can be addressed with no more than routineexperimentation. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to sound medical judgment.

Multiple doses of the molecules of the invention are also contemplated.In some instances, when the molecules of the invention are administeredwith another therapeutic, for instance, an anti-inflammatory agent, asub-therapeutic dosage of either the molecules or the other agent, or asub-therapeutic dosage of both, is used in the treatment of a subjecthaving, or at risk of developing a TNFα disorder. When the two classesof drugs are used together, the other agent may be administered in asub-therapeutic dose to produce a desirable therapeutic result. A“sub-therapeutic dose” as used herein refers to a dosage which is lessthan that dosage which would produce a therapeutic result in the subjectif administered in the absence of the other agent. Thus, thesub-therapeutic dose of a therapeutic agent is one which would notproduce the desired therapeutic result in the subject in the absence ofthe administration of the molecules of the invention. Therapeutic dosesof agents useful for treating TNFα disorders are well known in the fieldof medicine. These dosages have been extensively described in referencessuch as Remington's Pharmaceutical Sciences; as well as many othermedical references relied upon by the medical profession as guidance forthe treatment of infectious disease, cancer, and autoimmune disease.Therapeutic dosages of oligonucleotides have also been described in theart.

Dosing regimens may be several times a day, daily, every other day,weekly, biweekly any of the times there between or less frequently. Theterm “biweekly dosing” as used herein, refers to the time course ofadministering a substance (e.g., an anti-TNFα nucleic acid) to a subjectonce every two weeks. The oligonucleotides may be administered every7-20 days, every 11-17 days, or every 13-15 days, for example.

The oligonucleotides are administered in effective amounts. Theeffective amount of a compound of the invention in the treatment of adisease described herein may vary depending upon the specific compoundused, the mode of delivery of the compound, and whether it is used aloneor in combination. The effective amount for any particular applicationcan also vary depending on such factors as the disease being treated,the particular compound being administered, the size of the subject, orthe severity of the disease or condition. One of ordinary skill in theart can empirically determine the effective amount of a particularmolecule of the invention without necessitating undue experimentation.Combined with the teachings provided herein, by choosing among thevarious active compounds and weighing factors such as potency, relativebioavailability, patient body weight, severity of adverse side-effectsand preferred mode of administration, an effective prophylactic ortherapeutic treatment regimen can be planned which does not causesubstantial toxicity and yet is entirely effective to treat theparticular subject.

The oligonucleotides described herein can be used alone or in conjugateswith other molecules such as detection or cytotoxic agents in thedetection and treatment methods of the invention, as described in moredetail herein.

The oligonucleotide may be, for instance, coupled or conjugated to adetectable label. A detectable label is a moiety, the presence of whichcan be ascertained directly or indirectly. Generally, detection of thelabel involves an emission of energy by the label. The label can bedetected directly by its ability to emit and/or absorb photons or otheratomic particles of a particular wavelength (e.g., radioactivity,luminescence, optical or electron density, etc.). A label can bedetected indirectly by its ability to bind, recruit and, in some cases,cleave another moiety which itself may emit or absorb light of aparticular wavelength (e.g., epitope tag such as the FLAG epitope,enzyme tag such as horseradish peroxidase, etc.). An example of indirectdetection is the use of a first enzyme label which cleaves a substrateinto visible products. The label may be of a chemical, peptide ornucleic acid molecule nature although it is not so limited. Otherdetectable labels include radioactive isotopes such as P³² or H³,luminescent markers such as fluorochromes, optical or electron densitymarkers, etc., or epitope tags such as the FLAG epitope or the HAepitope, biotin, avidin, and enzyme tags such as horseradish peroxidase,β-galactosidase, etc. The label may be bound to an oligonucleotideduring or following its synthesis. There are many different labels andmethods of labeling known to those of ordinary skill in the art.Examples of the types of labels that can be used in the presentinvention include enzymes, radioisotopes, fluorescent compounds,colloidal metals, chemiluminescent compounds, and bioluminescentcompounds. Those of ordinary skill in the art will know of othersuitable labels for the oligonucleotides described herein, or will beable to ascertain such, using routine experimentation. Furthermore, thecoupling or conjugation of these labels to the oligonucleotides of theinvention can be performed using standard techniques common to those ofordinary skill in the art.

Conjugation of the oligonucleotides to a detectable label facilitates,among other things, the use of such agents in diagnostic assays. Anothercategory of detectable labels includes diagnostic and imaging labels(generally referred to as in vivo detectable labels) such as for examplemagnetic resonance imaging (MRI): Gd(DOTA); for nuclear medicine: ²⁰¹T1,gamma-emitting radionuclide 99mTc; for positron-emission tomography(PET): positron-emitting isotopes, (18)F-fluorodeoxyglucose ((18)FDG),(18)F-fluoride, copper-64, gadodiamide, and radioisotopes of Pb(II) suchas 203Pb; 111In. In such instances, the use of the oligonucleotide couldbe observed as the oligonucleotide provides an antisense effect.

The conjugations or modifications described herein employ routinechemistry, which chemistry does not form a part of the invention andwhich chemistry is well known to those skilled in the art of chemistry.The use of protecting groups and known linkers such as mono- andhetero-bifunctional linkers are well documented in the literature andwill not be repeated here.

As used herein, “conjugated” means two entities stably bound to oneanother by any physiochemical means. It is important that the nature ofthe attachment is such that it does not impair substantially theeffectiveness of either entity. Keeping these parameters in mind, anycovalent or non-covalent linkage known to those of ordinary skill in theart may be employed. In some embodiments, covalent linkage is preferred.Noncovalent conjugation includes hydrophobic interactions, ionicinteractions, high affinity interactions such as biotin-avidin andbiotin-streptavidin complexation and other affinity interactions. Suchmeans and methods of attachment are well known to those of ordinaryskill in the art. A variety of methods may be used to detect the label,depending on the nature of the label and other assay components.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more agents, dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. Moreover, for animal (e.g., human) administration, itwill be understood that preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards. The compounds are generally suitable foradministration to humans. This term requires that a compound orcomposition be nontoxic and sufficiently pure so that no furthermanipulation of the compound or composition is needed prior toadministration to humans.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences(1990), incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

The agent may comprise different types of carriers depending on whetherit is to be administered in solid, liquid or aerosol form, and whetherit need to be sterile for such routes of administration as injection.The present invention can be administered intravenously, intradermally,intraarterially, intralesionally, intratumorally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g., aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in creams, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, incorporated herein by reference).In a particular embodiment, intraperitoneal injection is contemplated.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more components. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The agent may be formulated into a composition in a free base, neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups also can be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

The compounds of the invention may be administered directly to a tissue.Direct tissue administration may be achieved by direct injection. Thecompounds may be administered once, or alternatively they may beadministered in a plurality of administrations. If administered multipletimes, the compounds may be administered via different routes. Forexample, the first (or the first few) administrations may be madedirectly into the affected tissue while later administrations may besystemic.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

According to the methods of the invention, the compound may beadministered in a pharmaceutical composition. In general, apharmaceutical composition comprises the compound of the invention and apharmaceutically-acceptable carrier. As used herein, apharmaceutically-acceptable carrier means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients.

Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers and other materials which arewell-known in the art. Such preparations may routinely contain salt,buffering agents, preservatives, compatible carriers, and optionallyother therapeutic agents. When used in medicine, the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically-acceptable saltsthereof and are not excluded from the scope of the invention. Suchpharmacologically and pharmaceutically-acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic, and the like. Also,pharmaceutically-acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts.

The compounds of the invention may be formulated into preparations insolid, semi-solid, liquid or gaseous forms such as tablets, capsules,powders, granules, ointments, solutions, depositories, inhalants andinjections, and usual ways for oral, parenteral or surgicaladministration. The invention also embraces pharmaceutical compositionswhich are formulated for local administration, such as by implants.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active agent. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. Techniques forpreparing aerosol delivery systems are well known to those of skill inthe art. Generally, such systems should utilize components which willnot significantly impair the biological properties of the active agent(see, for example, Sciarra and Cutie, “Aerosols,” in Remington'sPharmaceutical Sciences. Those of skill in the art can readily determinethe various parameters and conditions for producing aerosols withoutresort to undue experimentation.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

The compositions of the invention may be formulated in a topicalcomposition for administration to the skin or a body cavity. Suitabletopical vehicles and vehicle components are well known in the cosmeticand pharmaceutical arts, and include such vehicles (or vehiclecomponents) as water; organic solvents such as alcohols (particularlylower alcohols readily capable of evaporating from the skin such asethanol), glycols (such as propylene glycol, butylene glycol, andglycerin), aliphatic alcohols (such as lanolin); mixtures of water andorganic solvents (such as water and alcohol), and mixtures of organicsolvents such as alcohol and glycerin (optionally also with water);lipid-based materials such as fatty acids, acylglycerols (includingoils, such as mineral oil, and fats of natural or synthetic origin),phosphoglycerides, sphingolipids and waxes; protein-based materials suchas collagen and gelatin; silicone-based materials (both non-volatile andvolatile) such as cyclomethicone, demethiconol and dimethicone copolyol(Dow Corning); hydrocarbon-based materials such as petrolatum andsqualane; anionic, cationic and amphoteric surfactants and soaps;sustained-release vehicles such as microsponges and polymer matrices;stabilizing and suspending agents; emulsifying agents; and othervehicles and vehicle components that are suitable for administration tothe skin, as well as mixtures of topical vehicle components asidentified above or otherwise known to the art. The vehicle may furtherinclude components adapted to improve the stability or effectiveness ofthe applied formulation, such as preservatives, antioxidants, skinpenetration enhancers, sustained release materials, and the like.Examples of such vehicles and vehicle components are well known in theart and are described in such reference works as Martindale—The ExtraPharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.),Remington's Pharmaceutical Sciences.

The choice of a suitable vehicle will depend on the particular physicalform and mode of delivery that the formulation is to achieve. Examplesof suitable forms include liquids (e.g., gargles and mouthwashes,including dissolved forms of the strontium cation as well assuspensions, emulsions and the like); solids and semisolids such asgels, foams, pastes, creams, ointments, “sticks” (as in lipsticks orunderarm deodorant sticks), powders and the like; formulationscontaining liposomes or other delivery vesicles; rectal or vaginalsuppositories, creams, foams, gels or ointments; and other forms.Typical modes of delivery include application using the fingers;application using a physical applicator such as a cloth, tissue, swab,stick or brush (as achieved for example by soaking the applicator withthe formulation just prior to application, or by applying or adhering aprepared applicator already containing the formulation—such as a treatedor premoistened bandage, wipe, washcloth or stick—to the skin); spraying(including mist, aerosol or foam spraying); dropper application (as forexample with ear drops); sprinkling (as with a suitable powder form ofthe formulation); and soaking.

Topical formulations also include formulations for rectal and vaginaladministration. Formulations for rectal administration may be presentedas a suppository with a suitable base comprising, for example, cocoabutter. Formulations suitable for vaginal administration may bepresented as tablets, pessaries, tampons, creams, gels, pastes, foams orspray formulations containing in addition to the active ingredient suchcarriers as are known in the art to be appropriate.

In yet other embodiments, a delivery vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient. Other delivery systems can include time-release,delayed release or sustained release delivery systems. Such systems canavoid repeated administrations of the compound, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides.

In some embodiments the antisense nucleic acids of the invention areformulated as a stable self-assembling nanostructure. The nanostructureincludes a TNFα antisense oligonucleotide, wherein the antisenseoligonucleotide is associated with a core. The core may be a solid or ahollow core, such as a liposomal core. A solid core is a sphericalshaped material that does not have a hollow center. The term sphericalas used herein refers to a general shape and does not imply or is notlimited to a perfect sphere or round shape. It may includeimperfections.

Solid cores can be constructed from a wide variety of materials known tothose skilled in the art including but not limited to: noble metals(gold, silver), transition metals (iron, cobalt) and metal oxides(silica). In addition, these cores may be inert, paramagnetic, orsuperparamagnetic. These solid cores can be constructed from either purecompositions of described materials, or in combinations of mixtures ofany number of materials, or in layered compositions of materials. Inaddition, solid cores can be composed of a polymeric core such asamphiphilic block copolymers, hydrophobic polymers such as polystyrene,poly(lactic acid), poly(lactic co-glycolic acid), poly(glycolic acid),poly(caprolactone) and other biocompatible polymers known to thoseskilled in the art.

The core may alternatively be a hollow core, which has at least somespace in the center region of a shell material. Hollow cores includeliposomal cores. A liposomal core as used herein refers to a centrallylocated core compartment formed by a component of the lipids orphospholipids that form a lipid bilayer. “Liposomes” are artificial,self closed vesicular structure of various sizes and structures, whereone or several membranes encapsulate an aqueous core. Most typicallyliposome membranes are formed from lipid bilayers membranes, where thehydrophilic head groups are oriented towards the aqueous environment andthe lipid chains are embedded in the lipophilic core. Liposomes can beformed as well from other amphiphilic monomeric and polymeric molecules,such as polymers, like block copolymers, or polypeptides. Unilamellarvesicles are liposomes defined by a single membrane enclosing an aqueousspace. In contrast, oligo- or multilamellar vesicles are built up ofseveral membranes. Typically, the membranes are roughly 4 nm thick andare composed of amphiphilic lipids, such as phospholipids, of natural orsynthetic origin. Optionally, the membrane properties can be modified bythe incorporation of other lipids such as sterols or cholic acidderivatives.

The lipid bilayer is composed of two layers of lipid molecules. Eachlipid molecule in a layer is oriented substantially parallel to adjacentlipid bilayers, and two layers that form a bilayer have the polar endsof their molecules exposed to the aqueous phase and the non-polar endsadjacent to each other. The central aqueous region of the liposomal coremay be empty or filled fully or partially with water, an aqueousemulsion, oligonucleotides, or other therapeutic or diagnostic agents.

“Lipid” refers to its conventional sense as a generic term encompassingfats, lipids, alcohol-ether-soluble constituents of protoplasm, whichare insoluble in water. Lipids usually consist of a hydrophilic and ahydrophobic moiety. In water lipids can self organize to form bilayersmembranes, where the hydrophilic moieties (head groups) are orientedtowards the aqueous phase, and the lipophilic moieties (acyl chains) areembedded in the bilayers core. Lipids can comprise as well twohydrophilic moieties (bola amphiphiles). In that case, membranes may beformed from a single lipid layer, and not a bilayer. Typical examplesfor lipids in the current context are fats, fatty oils, essential oils,waxes, steroid, sterols, phospholipids, glycolipids, sulpholipids,aminolipids, chromolipids, and fatty acids. The term encompasses bothnaturally occurring and synthetic lipids. Preferred lipids in connectionwith the present invention are: steroids and sterol, particularlycholesterol, phospholipids, including phosphatidyl, phosphatidylcholinesand phosphatidylethanolamines and sphingomyelins. Where there are fattyacids, they could be about 12-24 carbon chains in length, containing upto 6 double bonds. The fatty acids are linked to the backbone, which maybe derived from glycerol. The fatty acids within one lipid can bedifferent (asymmetric), or there may be only 1 fatty acid chain present,e.g. lysolecithins. Mixed formulations are also possible, particularlywhen the non-cationic lipids are derived from natural sources, such aslecithins (phosphatidylcholines) purified from egg yolk, bovine heart,brain, liver or soybean.

The liposomal core can be constructed from one or more lipids known tothose in the art including but not limited to: sphingolipids such assphingosine, sphingosine phosphate, methylated sphingosines andsphinganines, ceramides, ceramide phosphates, 1-0 acyl ceramides,dihydroceramides, 2-hydroxy ceramides, sphingomyelin, glycosylatedsphingolipids, sulfatides, gangliosides, phosphosphingolipids, andphytosphingosines of various lengths and saturation states and theirderivatives, phospholipids such as phosphatidylcholines,lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids,cyclic LPA, phosphatidylethanolamines, lysophosphatidylethanolamines,phosphatidylglycerols, lysophosphatidylglycerols, phosphatidylserines,lysophosphatidylserines, phosphatidylinositols, inositol phosphates,LPI, cardiolipins, lysocardiolipins, bis(monoacylglycero) phosphates,(diacylglycero) phosphates, ether lipids, diphytanyl ether lipids, andplasmalogens of various lengths, saturation states, and theirderivatives, sterols such as cholesterol, desmosterol, stigmasterol,lanosterol, lathosterol, diosgenin, sitosterol, zymosterol, zymostenol,14-demethyl-lanosterol, cholesterol sulfate, DHEA, DHEA sulfate,14-demethyl-14-dehydrlanosterol, sitostanol, campesterol, ether anioniclipids, ether cationic lipids, lanthanide chelating lipids, A-ringsubstituted oxysterols, B-ring substituted oxysterols, D-ringsubstituted oxysterols, side-chain substituted oxysterols, doublesubstituted oxysterols, cholestanoic acid derivatives, fluorinatedsterols, fluorescent sterols, sulfonated sterols, phosphorylatedsterols, and polyunsaturated sterols of different lengths, saturationstates, and their derivatives.

The oligonucleotides may be positioned on the exterior of the core,within the walls of the core and/or in the center of the core. Anoligonucleotide that is positioned on the core is typically referred toas coupled to the core. Coupled may be direct or indirect. In someembodiments at least 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800,900 or 1,000 oligonucleotides or any range combination thereof are onthe exterior of the core. In some embodiments, 1-1000, 10-500, 50-250,or 50-300 oligonucleotides are present on the surface.

The oligonucleotides of the oligonucleotide shell may be oriented in avariety of directions. In some embodiments the oligonucleotides areoriented radially outwards. The orientation of these oligonucleotidescan be either 5′ distal/3′ terminal in relation to the core, or 3′distal/5′terminal in relation to the core, or laterally oriented aroundthe core. In one embodiment one or a multiplicity of differentoligonucleotides are present on the same surface of a single SNA. In allcases, at least 1 oligonucleotide is present on the surface but up to10,000 can be present.

The oligonucleotides may be linked to the core or to one another and/orto other molecules such an active agents either directly or indirectlythrough a linker. The oligonucleotides may be conjugated to a linker viathe 5′ end or the 3′ end, e.g. [Sequence, 5′-3′]-Linker orLinker-[Sequence, 5′-3′]. Some or all of the oligonucleotides of thenanostructure may be linked to one another either directly or indirectlythrough a covalent or non-covalent linkage. The linkage of oneoligonucleotide to another oligonucleotide may be in addition to oralternatively to the linkage of that oligonucleotide to liposomal core.

The oligonucleotide shell may be anchored to the surface of the corethrough one or multiple of linker molecules, including but not limitedto: any chemical structure containing one or multiple thiols, such asthe various chain length alkane thiols, cyclic dithiol, lipoic acid, orother thiol linkers known to those skilled in the art.

In an embodiment containing a liposomal core, the oligonucleotide shellmay be anchored to the surface of the liposomal core through conjugationto one or a multiplicity of linker molecules including but not limitedto: tocopherols, sphingolipids such as sphingosine, sphingosinephosphate, methylated sphingosines and sphinganines, ceramides, ceramidephosphates, 1-0 acyl ceramides, dihydroceramides, 2-hydroxy ceramides,sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides,phosphosphingolipids, and phytosphingosines of various lengths andsaturation states and their derivatives, phospholipids such asphosphatidylcholines, lysophosphatidylcholines, phosphatidic acids,lysophosphatidic acids, cyclic LPA, phosphatidylethanolamines,lysophosphatidylethanolamines, phosphatidylglycerols,lysophosphatidylglycerols, phosphatidylserines, lysophosphatidylserines,phosphatidylinositols, inositol phosphates, LPI, cardiolipins,lysocardiolipins, bis(monoacylglycero) phosphates, (diacylglycero)phosphates, ether lipids, diphytanyl ether lipids, and plasmalogens ofvarious lengths, saturation states, and their derivatives, sterols suchas cholesterol, desmosterol, stigmasterol, lanosterol, lathosterol,diosgenin, sitosterol, zymosterol, zymostenol, 14-demethyl-lanosterol,cholesterol sulfate, DHEA, DHEA sulfate,14-demethyl-14-dehydrlanosterol, sitostanol, campesterol, ether anioniclipids, ether cationic lipids, lanthanide chelating lipids, A-ringsubstituted oxysterols, B-ring substituted oxysterols, D-ringsubstituted oxysterols, side-chain substituted oxysterols, doublesubstituted oxysterols, cholestanoic acid derivatives, fluorinatedsterols, fluorescent sterols, sulfonated sterols, phosphorylatedsterols, and polyunsaturated sterols of different lengths, saturationstates, and their derivatives.

The oligonucleotide may also be associated with the core by beingembedded within the core (liposomal core) or it may be attached orlinked, either indirectly (i.e. non-covalently or covalently throughother molecules such a linkers) or directly (i.e. covalently).

The invention also includes articles, which refers to any one orcollection of components. In some embodiments the articles are kits. Thearticles include pharmaceutical or diagnostic grade compounds of theinvention in one or more containers. The article may includeinstructions or labels promoting or describing the use of the compoundsof the invention.

As used herein, “promoted” includes all methods of doing businessincluding methods of education, hospital and other clinical instruction,pharmaceutical industry activity including pharmaceutical sales, and anyadvertising or other promotional activity including written, oral andelectronic communication of any form, associated with compositions ofthe invention in connection with treatment of TNFα disorders.

“Instructions” can define a component of promotion, and typicallyinvolve written instructions on or associated with packaging ofcompositions of the invention. Instructions also can include any oral orelectronic instructions provided in any manner.

Thus the agents described herein may, in some embodiments, be assembledinto pharmaceutical or diagnostic or research kits to facilitate theiruse in therapeutic, diagnostic or research applications. A kit mayinclude one or more containers housing the components of the inventionand instructions for use. Specifically, such kits may include one ormore agents described herein, along with instructions describing theintended therapeutic application and the proper administration of theseagents. In certain embodiments agents in a kit may be in apharmaceutical formulation and dosage suitable for a particularapplication and for a method of administration of the agents.

The kit may be designed to facilitate use of the methods describedherein by physicians and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for human administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container.

The kit may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag. The kit may besterilized after the accessories are added, thereby allowing theindividual accessories in the container to be otherwise unwrapped. Thekits can be sterilized using any appropriate sterilization techniques,such as radiation sterilization, heat sterilization, or othersterilization methods known in the art. The kit may also include othercomponents, depending on the specific application, for example,containers, cell media, salts, buffers, reagents, syringes, needles, afabric, such as gauze, for applying or removing a disinfecting agent,disposable gloves, a support for the agents prior to administration etc.

The compositions of the kit may be provided as any suitable form, forexample, as liquid solutions or as dried powders. When the compositionprovided is a dry powder, the powder may be reconstituted by theaddition of a suitable solvent, which may also be provided. Inembodiments where liquid forms of the composition are sued, the liquidform may be concentrated or ready to use. The solvent will depend on thecompound and the mode of use or administration. Suitable solvents fordrug compositions are well known and are available in the literature.The solvent will depend on the compound and the mode of use oradministration.

The kits, in one set of embodiments, may comprise a carrier means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method. Forexample, one of the containers may comprise a positive control for anassay. Additionally, the kit may include containers for othercomponents, for example, buffers useful in the assay.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. In thecase of dosage forms suitable for parenteral administration the activeingredient is sterile and suitable for administration as a particulatefree solution. In other words, the invention encompasses both parenteralsolutions and lyophilized powders, each being sterile, and the latterbeing suitable for reconstitution prior to injection. Alternatively, theunit dosage form may be a solid suitable for oral, transdermal, topicalor mucosal delivery.

In a preferred embodiment, the unit dosage form is suitable forintravenous, intramuscular or subcutaneous delivery. Thus, the inventionencompasses solutions, preferably sterile, suitable for each deliveryroute.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat the TNFαdisease or disorder. In other words, the article of manufacture includesinstruction means indicating or suggesting a dosing regimen including,but not limited to, actual doses, monitoring procedures and othermonitoring information.

More specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material. The invention also provides anarticle of manufacture comprising packaging material, such as a box,bottle, tube, vial, container, sprayer, insufflator, intravenous (i.v.)bag, envelope and the like; and at least one unit dosage form of eachpharmaceutical agent contained within said packaging material. Theinvention further provides an article of manufacture comprisingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of each pharmaceutical agent containedwithin said packaging material. The invention further provides anarticle of manufacture comprising a needle or syringe, preferablypackaged in sterile form, for injection of the formulation, and/or apackaged alcohol pad.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

EXAMPLES Example 1: Inhibitory Oligonucleotide Sequences

Oligonucleotides were synthesized on a MerMade 48 (Bioautomation) usingstandard phosphoramidite chemistry (reagents from Glen Research andChemGenes). Three of the sequences were targeted to the start codon ofhuman TNF mRNA (Oligos 3482, 3483, and 3484). Three additional sequenceswere generated with Sfold (Oligos 3485, 3486, and 3487). One sequence,Oligo 3488, was humanized. Finally, a scrambled control was generated(Oligo 3495). The respective sequences are listed in Table 1:

TABLE 1 List of Oligonucleotide Sequences Oligo Sequence SEQ Oligo ID(5′ to 3′) ID NO. Oligo 3482 CATGGTGTCCTTTCCAGG 1 Oligo 3483TCAGTGCTCATGGTGTCC 2 Oligo 3484 CATGCTTTCAGTGCTCAT 3 Oligo 3485TGGGAGTAGATGAGGTAC 4 Oligo 3486 TTGACCTTGGTCTGGTAG 5 Oligo 3487GATGGCAGAGAGGAGGTT 6 Oligo 3488 TTATCTCTCAGCTCCACG 7 Oligo 3495ATGGAGCAAAACCCGCAG 8

The oligonucleotides were then purified with reverse phase highperformance liquid chromatography (Aglient). The oligonucleotide productidentity was verified with matrix-assisted laser desorption ionizationmass spectrometry.

The synthesized oligonucleotides were initially screened for TNFexpression. Primary human keratinocytes were plated in 96 well plates.The following day, the cells were transfected with 2 μM of the antisensestrand using lipofectamine (Life Technologies) in Optimem (LifeTechnologies) for 24 hours. After the incubation period, the cells werewashed with PBS, their mRNA extracted, cDNA synthesized, and TNF andGAPDH levels were determined with RT-PCR (FIG. 1). Oligonucleotides3482, 4385 and 3487 were particularly effective at decreasing TNFexpression levels.

The same experimental conditions were performed in an additional set ofmodified sequences. In brief, primary human keratinocytes were plated in96 well plates. The following day, the cells were transfected with 2 μMof the antisense strand using lipofectamine (Life Technologies) inOptimem (Life Technologies) for 24 hours. After the incubation period,the cells were washed with PBS, their mRNA extracted, cDNA synthesized,and TNF and GAPDH levels were probed with RT-PCR (FIG. 2). Each sequencewas paired with a respective control (italicized), as seen in Table 2:

TABLE 2 Modified Oligonucleotide Sequences and their ControlsOligo Sequence (5′ to 3′) (m = methylated; * = SEQ Oligo ID Oligo Namephosphorothioate linkage) ID NO. Oligo 3485 hTNF 568-POTGGGAGTAGATGAGGTAC  4 Oligo 3495 scr-18 PO ATGGAGCAAAACCCGCAG  8Oligo 3742 hTNF 568-OmePO mUmGmGmGmAmGmUmAmGmAmUmG 10 mAmGmGmUmAmCOligo 3508 scr-18 OMePO mAmUmGmGmAmGmCmAmAmAmAmC 11 mCmCmGmCmAmGOligo 3500 hTNF 568-PS T*G*G*G*A*G*T*A*G*A*T*G* 12 A*G*G*T*A*COligo 3496 scr-18 PS A*T*G*G*A*G*C*A*A*A*A*C* 13 C*C*G*C*A*G Oligo 3526hTNF 568-OmePS mU*mG*mG*mG*mA*mG*mU*mA* 14 mG*mA*mU*mG*mA*mG*mG*mU*mA*mC Oligo 3607 scr-18 OMePS mA*mU*mG*mG*mA*mG*mC*mA* 15mA*mA*mA*mC*mC*mC*mG*mC* mA*mG Oligo 3534 hTNF 568-mUmGmGmGmAmGT*A*G*A*T*G* 16 Gapmer/OmePO-PS mAmGmGmUmAmC Oligo 3514scr-18 mAmUmGmGmAmGC*A*A*A*A*C* 17 gapmer/OMePO-PS mCmCmGmCmAmGOligo 3509 hTNF 568- mU*mG*mG*mG*mA*mG*T*A*G* 18 Gapmer/OmePS-PSA*T*G*mA*mG*mG*mU*mA*mC Oligo 3513 scr-18 mA*mU*mG*mG*mA*mG*C*A*A* 19gapmer/OMePS-PS A*A*C*mC*mC*mG*mC*mA*mG

Example 2: Inhibition of TNF by Spherical Nucleic Acids (SNAs)

The chemistries of different SNAs were compared with respectiveoligonucleotide controls. SNAs containing anti-TNF antisense strandswere prepared. 13±1 nm diameter gold nanoparticles were prepared byreducing a 490 mL boiling aqueous solution of 0.1969 g of HAuCl4⋅3 H2Owith 0.570 g of trisodium citrate in 10 mL of water. The particlesolution was then filtered through a 0.45 μm cellulose acetate membraneto remove any aggregated nanoparticles. The nanoparticle concentrationwas 11 nM as prepared.

The gold nanoparticles were then used as is to prepare anti-TNF andcontrol SNAs. The SNA synthesis began by adding a thiolated 5 kDa linearpoly(ethylene glycol) to the as-synthesized gold nanoparticles to afinal concentration of 5 μM. After mixing, the solution was allowed tostand for 1.5 hours at 37° C., at which point the oligonucleotides wereadded to the solution at a final concentration of 5 μM. Whilemaintaining the temperature, a solution of sodium chloride was added tothe functionalization mixture in two equal aliquots over the course of 1hour to raise the concentration of NaCl to 150 mM. That mixture wasallowed to stand at 37° C. overnight. The following day, centrifugationat 21,000×g precipitated the particles, the supernatant was removed, andthe particles were resuspended in sterile PBS. This process was repeatedthree times to remove excess PEG and oligonucleotide that had notadhered to the particles. The number of oligonucleotides pernanoparticles was measured by liberating the oligonucleotides from thegold core—the nanoparticles were oxidatively dissolved with KCN.Finally, the number of oligonucleotides liberated was measured with afluorescence based assay (Oligreen, Life Technologies) according to themanufacturer's instructions.

Primary human keratinocytes were plated in 96 well plates. The followingday, the cells were transfected with the TNF or control SNAs at anoligonucleotide concentration of 5 μM. The treatment was allowed toproceed overnight. The following day, the cells were washed, the mRNAcollected, cDNA prepared, and the expressions of TNF and GAPDH wereprobed. The results are shown in FIG. 3. Each sequence was paired with arespective control (italicized) as seen in Table 3:

TABLE 3 Oligonucleotide Sequences and ControlsUsed to Compare SNA Chemistries Oligo Sequence (5′ to 3′)(m = methylated; * =  SEQ Oligo ID Oligo Name phosphorothioate linkage)ID NO. Oligo 3652 hTNF568-Gapmer- mU*mG*mG*mG*mA*mG*T*A*G*A*T*G*mA* 20OMePS/PS-SH mG*mG*mU*mA*mC*/iSp18//iSp18// 3ThioMC3-D/ Oligo 4030mA*mU*mG*mG*mA*mG*C*A*A*A*A*C*mC* 21 mC*mG*mC*mA*Mg/iSp18//iSp18//3ThioMC3-D/ Oligo 3657 hTNF568-Gapmer- mUmGmGmGmAmGT*A*G*A*T*G*mAmGmG 22OMePO/PS-SH mUmAmC/iSp18//iSp18//3ThioMC3-D/ Oligo 4028mAmUmGmGmAmGC*A*A*A*A*C*mCmCmG 23 mCmAmG/iSp18//iSp18//3ThioMC3-D/Oligo 3743 hTNF568-PO-SH TGGGAGTAGATGAGGTAC/iSp18//iSp18// 243ThioMC3-D/ Oligo 3660 ATGGAGCAAAACCCGCAG/iSp18//iSp18// 25 3ThioMC3-D/

Human keratinocytes were plated in 96 well tissue culture plates andallowed to adhere overnight. The next day, they were treated with 50ng/mL human recombinant TNF for 4 hours prior to treatment with Oligo3661 modified SNAs or Oligo 3657 (SEQ ID NO:22) SNAs. The treatment wasallowed to proceed overnight. The following day the cells were washed,the mRNA collected, cDNA prepared, and the expression of TNF and GAPDHwere probed and the percent gene knockdown was calculated (FIG. 4).

The effect of additional phosphorothioate modifications on Oligo3657-modified SNAs was examined. A family of oligonucleotides withincreasing phosphorothioate content was prepared as using goldnanoparticles as previously described. The particles were transfectedinto human keratinocytes overnight. The following day, the cells werewashed, the mRNA collected, cDNA prepared, and the expression of TNF andGAPDH were probed and the percent gene knockdown was calculated (FIG.5). The sequences in Table 4 were analyzed:

TABLE 4 Oligonucleotides with Increasing Phosphorothioate ContentOligo Sequence (5′ to 3′) (m = methylated; * =  SEQ Oligo ID Oligo Namephosphorothioate linkage) ID NO. Oligo 5196 hTNF 568-1 PSmUmGmGmGmAmG*T*A*G*A*T*G*mAmGmGmU 26 mAmC/isp18//isp18//3thiomc3-d/Oligo 5289 hTNF 568-2 PS mUmGmGmGmA*mG*T*A*G*A*T*G*mAmGmGmU 27mAmC/isp18//isp18//3thiomc3-d/ Oligo 5290 hTNF 568-3 PSmUmGmGmG*mA*mG*T*A*G*A*T*G*mAmGmGmU 28 mAmC/isp18//isp18//3thiomc3-d/Oligo 5291 hTNF 568-4 PS mUmGmG*mG*mA*mG*T*A*G*A*T*G*mAmGmG 29mUmAmC/isp18//isp18//3thiomc3-d/ Oligo 5292 hTNF 568-5 PSmUmG*mG*mG*mA*mG*T*A*G*A*T*G*mAmGmG 30 mUmAmC/isp18//isp18//3thiomc3-d/Oligo 5293 hTNF 568-6 PS mU*mG*mG*mG*mA*mG*T*A*G*A*T*G*mAmGmG 31mUmAmC/isp18//isp18//3thiomc3-d/

The effect of hollow SNAs on TNF expression was examined.Oligonucleotides containing a tocopherol phosphoramidite were prepared(reagents from ChemGenes). Liposomal scaffolds consisting ofdioleoylphosphatidyl-choline (DOPC) were prepared by dissolving thelipid in DCM at 75 mg/mL, and then the solution dried under a stream ofnitrogen and lyophilized. The residual material was dissolved in HEPES(pH 7.3) and 150 mM NaCl at a concentration of 40 mg/mL, and allowed tostand for 30 minutes, followed by three freeze/thaw cycles using liquidnitrogen. That solution was then extruded through polycarbonatemembranes containing pores with diameters of 100, 50, and 30 nm. Theresultant solution contained liposomes approximately 40 nm in diameter.Functionalization into SNAs occurred by adding the tocopherol-modifiedoligonucleotides to the liposome solution. The materials were thenconcentrated with tangential flow filtration and applied to humankeratinocytes as described above. The sequences prepared were as inTable 5:

TABLE 5 Oligonucleotide Sequence and itsControl for Hollow SNA Investigation Oligo Sequence (5′ to 3′) Oligo(m = methylated; * =  SEQ Oligo ID Name phosphorothioate linkage) ID NO568T TNF568- mUmGmGmGmAmGT*A*G*A*T*G* 32 toco mAmGmGmUmAmC/iSp18//iSp18//toco/ ControlT Control- mAmUmGmGmAmGC*A*A*A*A*C* 33 tocomCmCmGmCmAmG/iSp18// iSp18//toco/

Example 3: The Inhibitory Effect of Antisense SNAs Targeting TNF mRNA

The inhibitory effect of antisense SNAs targeting TNF mRNA on mRNAexpression was compared to non-targeting control SNAs. SNAs containinganti-TNF antisense strands, composed of compound 6081, were prepared.

All oligonucleotides were synthesized at the 1 μmole scale employingstandard UniLinker (ChemGenes). The DNA, RNA, 2′-O-Me monomers andhexa(ethylene glycol) spacers were obtained from ChemGenes Corporation.The cholesterol modifier was obtained from Glen Research. Linkages wereeither standard phosphodiesters or phosphorothioates made with 0.2 Mphenylacetyl disulfide (PADS) in 1:1 lutidine:ACN as the sulfurizationagent. Synthesis was performed DMT-off, in the 5′ to 3′ direction. Aftersynthesis, the oligonucleotides were cleaved from the support andde-protected using a 4:1 mixture of ammonium hydroxide and ethanol at55° C. for 16 hours. The oligonucleotides were purified via ion-exchangehigh performance liquid chromatography (HPLC) techniques. Molecularweights and extinction coefficients were estimated using IDTOligoAnalyzer. Verification of oligonucleotide molecular weight wasperformed using matrix-assisted laser desorption/ionization (MALDI).Oligonucleotide concentration was determined by UV-absorbance at 260 nmon a microplate reader (BioTek) together with the calculated extinctioncoefficient from the IDT OligoAnalyzer.

The oligonucleotides were then used to prepare anti-TNF and controlsSNAs. The synthesis began by diluting the oligonucleotides to 100 μM inPBS. The oligonucleotides were then stored overnight, protected fromlight, at 4° C. Due to the electrostatic repulsion of the polar solventtowards the hydrophobic cholesterol tail of the oligonucleotide,structured micelle structures form with a cholesterol core andoligonucleotides extending outward.

Primary human keratinocytes were plated in 96 well plates. The followingday, the cells were treated with 50 ng/mL human recombinant TNF for 4hours prior to being transfected with Oligo ID 6081 or Oligo ID 6093SNAs at oligonucleotide concentrations of 1000, 333.3, 100, 33.3, 10,3.3, 1, 0.33, 0.1, 0.03 and 0.01 nM. The treatment was allowed toproceed overnight. The following day, the cells were washed, the mRNAcollected, cDNA prepared, and the expressions of TNF and GAPDH wereprobed and the percent gene expression was calculated (FIG. 9).

The effect of varying the chain lengths of hexaethylene glycol (HEG) onthe antisense activity of Oligo ID 6081 SNAs was examined.Oligonucleotides with increasing HEG spacer lengths were prepared andformulated into self-assembling SNAs. Primary human keratinocytes wereplated in 96 well plates. The following day, the cells were treated with50 ng/mL human recombinant TNF for 4 hours prior to being transfectedwith Oligo ID 6080, 6081, 6082, 6092, 6083, 6093, 6094 or 6095 SNAs atoligonucleotide concentrations of 100, 10, 1, and 0.1 nM. The treatmentwas allowed to proceed overnight. The following day, the cells werewashed, the mRNA collected, cDNA prepared, and the expressions of TNFand GAPDH were probed and the percent gene expression was calculated(FIG. 10).

TABLE 6 Oligonucleotide Sequences for Hollow SNA Investigation Oligo SEQID Oligonucleotide Sequence (5′ to 3′) ID NO. 6080mUmGmGmGmAmGT*A*G*A*T*G*mAmGmGmUmAmC/ 35 iSp18//iSp18//iSp18//3CholTEG/6081 mUmGmGmGmAmGT*A*G*A*T*G*mAmGmGmUmAmC/ 36 iSp18//iSp18//3CholTEG/6082 mUmGmGmGmAmGT*A*G*A*T*G*mAmGmGmUmAmC/ 37 iSp18//3CholTEG/ 6092mUmGmGmGmAmGT*A*G*A*T*G*mAmGmGmUmAmC/ 38 3CholTEG/ 6083mAmUmGmGmAmGC*A*A*A*A*C*mCmCmGmCmAmG/ 39 iSp18//iSp18//iSp18//3CholTEG/6093 mAmUmGmGmAmGC*A*A*A*A*C*mCmCmGmCmAmG/ 40 iSp18//iSp18//3CholTEG/6094 mAmUmGmGmAmGC*A*A*A*A*C*mCmCmGmCmAmG/ 41 iSp18//3CholTEG/ 6095mAmUmGmGmAmGC*A*A*A*A*C*mCmCmGmCmAmG/ 42 3CholTEG/ “*” denotes aphosphorotioate bond, “m” denotes an O'methylated base,“/iSp18/” denotes a hexa(ethylene glycol) spacer, “/3CholTEG/” denotes a3′ tri(ethylene glycol) bound to a cholesterol

Example 4: Testing Ex Vivo Activity in Human Psoriatic Skin

Oligonucleotides were synthesized with a Mermade 48 (Bioautomation)using standard solid phase phosphoramidite methodology. Bases andreagents were purchased from Glen Research and Chemgenes. Alloligonucleotides were purified by reverse-phase high performance liquidchromatography (HPLC) and molecular weights were measured usingmatrix-assisted laser desorption/ionization (MALDI) analysis. Thesynthesized oligonucleotides are listed in Table 7.

Liposomes were synthesized by extrusion of1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) hydrated in phosphatebuffered saline solution (PBS) (137 mM NaCl, 10 M phosphate, 2.7 mM KCl,pH 7.4, hyclone) using 47 mm diameter polycarbonate membranes with 50 nmpores (Sterlitech). Liposome diameters were measured using dynamic lightscattering using a Malvern Zetasizer Nano (Malvern Instruments). Lipidconcentration was determined using a phospholipid assay kit (Sigma).

L-SNAs were synthesized by mixing oligonucleotides to liposomes in a100:1 molar ratio, and incubating the mixture at room temperature for 4hours. L-SNAs were isolated from unreacted materials and concentratedusing tangential flow filtration (TFF) using a MiniKros Pilot i System(Spectrum Labs) with a 30 kD molecular weight cutoff modifiedpolyethersulfone (mPES) hollow fiber filter module. Retentate containingL-SNAs were analyzed for lipid concentration using a phospholipid assaykit (Sigma). L-SNAs were diluted in 90% methanol to dissolve liposomesto release surface functionalized oligonucleotides whose concentrationwas measured using a Cary UV/vis spectrophotometer (Agilent). Theaverage number of oligonucleotides conjugated to a nanoparticle wascalculated by dividing the concentration of oligonucleotides by theconcentration of liposomes. L-SNA diameters were measured using dynamiclight scattering using a Malvern Zetasizer Nano (Malvern Instruments).

The gene regulatory effects of applying L-SNAs containing Oligo 4831.1on human psoriatic ex vivo skin cultures was examined. Experimentalgroups are listed in Table 8.

Four 3 mm diameter skin biopsies were taken from psoriatic plaques of 5different patients with mild to moderate plaque psoriasis. Eachreplicate was taken from a different patient for accurate representationand against a patient specific response. Explants were cultured in thefollowing manner. Holes were punched into the center of Corning 12-wellcell culture filter inserts. The dermal (bottom) portion of the explantswere pushed into the hole in the filter such that the biopsy wasembedded in the filter. The epidermis was exposed in the inner chamber,and the dermis was exposed in the outer chamber. These filters wereimmersed in 1 ml cell culture medium (DMEM, 1% FBS, 1.25 μg/mLamphotericin B, 50 μg/mL gentamicin, 0.1 U/mL penicillin/streptomycin)such that the dermis contacted the medium in the well and the epidermiswas exposed to air. Cultures were treated in the following manner: 2 μLof each compound was applied in PBS to the top (stratum corneum) of eachbiopsy using a pipette. Biopsies were incubated for 16 hours at 37° C.

Samples were homogenized immediately in 300 μL of RLT buffer (Qiagen) in2 mL screw cap vials filled with 3 mm diameter zirconia ball bearingsusing a ball bearing homogenizer. Homogenized samples were immediatelyflash frozen and stored at −80 ° C. Lysates were processed using aQiagen 96 well RNeasy plate extraction kit. RTPCR was performed on aLightcycler (Roche Diagnostics) using probes and primers against hTNFand hGAPDH (Roche Diagnostics) and TNF gene expression was measured(FIG. 11).

TABLE 7 Oligonucleotide sequences SEQ Name Oligo ID Sequence (5′ to 3′)ID NO. Control Oligo4832.1 mGmUmUmUmCmAC*C*A*C*C* 43C*mAmAmUmUmCmC/iSp18// iSp18//3Toco/ TNF Oligo4831.1mUmGmGmGmAmGT*A*G*A*C* 44 Anti- A*mAmGmGmUmAmC/iSp18// senseiSp18//3Toco/ Special bases used in the oligonucleotides are as follows:mX = 2′ O-methyl RNA, /iSp18/ = Spacer18 phosphoramidite, /3Toco/= 3′ tocopherol, * = phosphorothioate

TABLE 8 Ex Vivo psoriatic skin groups and treatments Tissue GroupsSource Oligo Target Type Concentration 1 Patient A Oligo 4832.1 ControlL-SNA 100 μM 2 Patient A Oligo 4831.1 TNF L-SNA 100 μM 3 Patient A Oligo4831.1 TNF L-SNA  10 μM 4 Patient A Oligo 4831.1 TNF L-SNA  1 μM 5Patient B Oligo 4832.1 Control L-SNA 100 μM 6 Patient B Oligo 4831.1 TNFL-SNA 100 μM 7 Patient B Oligo 4831.1 TNF L-SNA  10 μM 8 Patient B Oligo4831.1 TNF L-SNA  1 μM

Example 5: Testing the Efficacy of Anti-TNF SNAs and L-SNAs inTNBS-Induced Inflammatory Bowel Disease (IBD) Mouse Model

The effect of Oligo 227901 (Table 9) was assessed in a TNBS-induced IBDmice model. Oligo 227901 was synthesized with cholesterol at its 3′end(reagents from ChemGenes). In example 6, we show the formation ofself-assembled SNA structure of this oligonucleotide using Cryo-EM. Inaddition, an L-SNA form of Oligo 227901 has been prepared by addingoligonucleotide to 50 nm DOPC liposome solutions. S-SNAs and L-SNAs werebrought to pH 9.5 in bicarbonate solution for using in animals.

Studies of TNBS-induced colitis were performed in 6-7 weeks old Balb/cmice. For the induction of colitis, 10 mg of TNBS dissolved in 80%ethanol was administered per animal intra rectally on study day 0.Controls mice consisted of mice treated with vehicle only and untreatednaïve mice.

To examine therapeutic effect of S-SNAs and L-SNA of Oligo 227901 onTNBS-induced colitis study, mice were treated with compounds on day 1,2, 3 and 4 (total 4 doses) from 100 μg/dose/mice to 300 μg/dose/mice byoral gavage after the induction of colitis. The mice were monitoreddaily up to 7 days for clinical score observations, and euthanized onday 7 for analysis of gross pathology.

Clinical scores for the control mice and treated mice were assigned byconsidering the parameters: body weight, stool consistency and bleedingper rectum and any abnormalities observed in fur coat and abdomen.

Gross pathology scores were assigned to the control and treated mice onthe last day of study from the colons removed from the animals aftereuthanization. Gross pathology scores from 0 to 5 were assigned based onthe observations: No abnormalities detected (score 0); edema and rednessin one location (score 1); edema and redness in more than one location(score 2); One ulcer (score 3); more than one ulcer or severe ulcer(score 4); and edema and redness in more than one location, and one ormore than one ulcer (score 5).

The results of the study are represented in FIG. 12. The statisticalsignificance of the groups was calculated using one way ANOVA followedby Tukey's post-hoc test. An increase in clinical score and grosspathology score in the TNBS-only group suggests that rectaladministration of TNBS established colitis disease in all of the groupsexcept naïve mice. A significant reduction in the mean clinical scorecompared to the relevant vehicle group was observed for the grouptreated with S-SNA form of Oligo 227901 at an amount 200 μg/dose oftotal 4 doses from day 1 to day 4 after colitis induction with TNBS onday 0. Animals that were treated from day 1 until day 4 with four dosesof S-SNA (200 μg/dose and 300 μg/dose) and L-SNA (200 μg/dose) showedsignificant reduction in gross pathology score compared to vehiclegroup. Overall, the results suggest that oral administration of S-SNAhad a positive effect on disease symptoms reflecting lower clinicalscore, lower pathology score and higher animal survival rate.

TABLE 9 Oligonucleotide Sequence in InflammatoryBowel Disease (IBD) Investigation Oligo Sequence (5′ to 3′) Oligo(m = methylated; * = SEQ ID Oligo Name phosphorothioate linkage) ID NO.227901 TNF568-chol mUmGmGmGmAmGT*A*G*A*T*G* 45 mAmGmGmUmAmC/iSp18//iSp18//chol/

Example 6: Cryo-TEM of Self-Assembling SNA

CryoTEM imaging was performed on a solution of Oligo6307 Self-AssemblingSpherical Nucleic Acids (S-SNAs) (Table 10). S-SNA were prepared inphosphate buffered saline solution (PBS) (137 mM NaCl, 10 M phosphate,2.7 mM KCl, pH 7.4, Hyclone) using a 2.4 mM oligonucleotide solution.The sample was preserved in vitrified ice supported by holey carbonfilms on 400-mesh copper grids. Each sample was prepared by applying a 3μl drop of sample suspension to a cleaned grid, blotting away withfilter paper, and immediately proceeding with vitrification in liquidethane. Grids were stored under liquid nitrogen until transferred to theelectron microscope for imaging.

Electron microscopy was performed using an FEI Tecnai T12 electronmicroscope, operating at 120 keV equipped with an FEI Eagle 4K×4 k CCDcamera. Vitreous ice grids were transferred into the electron microscopeusing a cryostage that maintains the grids at a temperature below −170°C. High magnification image was acquired at a nominal magnification of110,000× (0.10 nm/pixel). Image was acquired at a nominal underfocus of−5 μm to −3 μm and electron doses of 10 to 40 e-/A².

The sample is primarily composed of many small ˜3 nm round particlesthroughout the field (FIG. 13). These round particles correspond to thediameter of a cluster of cholesterol molecules that make up hydrophobiccore of the S-SNA particle. The small ˜3 nm round particles are evenlyspaced by 3 to 5 nm gaps which likely correspond to the oligonucleotidecorona arrayed in a spherical orientation around the hydrophobic core(FIG. 14A). The oligonucleotide corona or shell defines the hydrodynamicradius of the S-SNA which is approximately 13 nm, resulting in the evenspacing between the electron dense hydrophobic cores (FIG. 14B).

TABLE 10 Oligonucleotide Sequence SEQ ID Oligo ID Sequence (5′ to 3′)NO. Oligo 6307 mUmGmGmGmAmG T*A*G*A*C*A* 46 mAmGmGmUmAmC/iSp18//iSp18//3Chol/

What is claimed is:
 1. A compound comprising the structure depicted inFIG. 7 or salts thereof.
 2. The compound of claim 1, wherein thecompound is 18 nucleotides in length.
 3. The compound of claim 1,formulated in a composition with a carrier.
 4. The compound of claim 1,wherein the compound is a sodium salt.
 5. The compound of claim 1,wherein the compound is the structure depicted in FIG.
 8. 6. Thecompound of any one of claims 1-5, wherein the compound furthercomprises a molecular species at one of the ends.
 7. The compound of anyone of claims 1-5, wherein the compound further comprises a molecularspecies at both ends.
 8. The compound of any one of claims 6-7, whereinthe molecular species is selected from the group consisting of a spacer,a lipid, a sterol, cholesterol, stearyl, C16 alkyl chain, bile acids,cholic acid, taurocholic acid, deoxycholate, oleyl litocholic acid,oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids,isoprenoids, such as steroids, vitamins, such as vitamin E, saturatedfatty acids, unsaturated fatty acids, fatty acid esters, such astriglycerides, pyrenes, porphyrines, Texaphyrine, adamantane, acridines,biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin,dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyaninedyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, and ibuprofen. 9.The compound of any one of claims 6-7, wherein the molecular species isa selected from the group consisting of a lipophilic moiety; a folicacid radical; a steroid radical; a carbohydrate radical; a vitamin Aradical; a vitamin E radical; or a vitamin K radical.
 10. The compoundof any one of claims 6-9, wherein the molecular species is connecteddirectly to the compound through a linkage selected from the groupconsisting of phosphodiester, phosphorothioate, methylphosphonate, andamide linkages.
 11. The compound of any one of claims 6-9, wherein themolecular species is connected indirectly to the compound through alinker.
 12. The compound of claim 11, wherein the linker is anon-nucleotidic linker selected from the group consisting of abasicresidues (dSpacer), oligoethyleneglycol, such as triethyleneglycol(spacer 9) or hexaethylenegylcol (spacer 18), and alkane-diol, such asbutanediol.
 13. An oligonucleotide comprisingmUmGmGmGmAmGT*A*G*A*T*G*mAmGmGmUmAmC (SEQ ID NO. 16), wherein theoligonucleotide is 18 nucleotides in length, wherein m is a 2′O methyl,and wherein * is a phosphorothioate modification.
 14. Theoligonucleotide of claim 13, wherein the oligonucleotide is formulatedin a composition with a carrier.
 15. The oligonucleotide of claim 13,wherein the carrier is a lipid based carrier.
 16. The oligonucleotide ofclaim 13, wherein the carrier is a nanoparticle.
 17. The oligonucleotideof any one of claims 13-16, wherein the oligonucleotide furthercomprises a molecular species at the 3′ or 5′ end.
 18. Theoligonucleotide of any one of claims 13-16, wherein the oligonucleotidefurther comprises a molecular species at both the 3′ and 5′ ends. 19.The oligonucleotide of any one of claims 17-18, wherein the molecularspecies is selected from the group consisting of a spacer, a lipid, asterol, cholesterol, stearyl, C16 alkyl chain, bile acids, cholic acid,taurocholic acid, deoxycholate, oleyl litocholic acid, oleoyl cholenicacid, glycolipids, phospholipids, sphingolipids, isoprenoids, such assteroids, vitamins, such as vitamin E, saturated fatty acids,unsaturated fatty acids, fatty acid esters, such as triglycerides,pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin,coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin,dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyaninedyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, and ibuprofen. 20.The oligonucleotide of any one of claims 17-18, wherein the molecularspecies is a selected from the group consisting of a lipophilic moiety;a folic acid radical; a steroid radical; a carbohydrate radical; avitamin A radical; a vitamin E radical; or a vitamin K radical.
 21. Theoligonucleotide of any one of claims 17-18, wherein the molecularspecies is connected directly to the compound through a linkage selectedfrom the group consisting of phosphodiester, phosphorothioate,methylphosphonate, and amide linkages.
 22. The oligonucleotide of anyone of claims 17-20, wherein the molecular species is connectedindirectly to the compound through a linker.
 23. The oligonucleotide ofclaim 22, wherein the linker is a non-nucleotidic linker selected fromthe group consisting of abasic residues (dSpacer), oligoethyleneglycol,such as triethyleneglycol (spacer 9) or hexaethylenegylcol (spacer 18),and alkane-diol, such as butanediol.
 24. An oligonucleotide comprising5′ TGGGAGTAGATGAGGTAC 3′ (SEQ ID NO. 4), wherein the oligonucleotide is18-19 nucleotides in length, wherein 4-6 nucleotides at the 5′ end and4-6 nucleotides at the 3′ end of the oligonucleotide include a 2′Omethyl, and wherein 4-10 nucleotides have a phosphorothioatemodification.
 25. The oligonucleotide of claim 24, wherein the 6nucleotides at the 5′ end and 6 nucleotides at the 3′ end of theoligonucleotide include a 2′O methyl.
 26. The oligonucleotide of claim24 or 25, wherein 6 nucleotides have a phosphorothioate modification.27. The oligonucleotide of claim 24 or 25, wherein 7 nucleotides have aphosphorothioate modification.
 28. The oligonucleotide of claim 24 or25, wherein 8 nucleotides have a phosphorothioate modification.
 29. Theoligonucleotide of any one of claims 24-28, wherein the phosphorothioatemodified nucleotides are in a central region of the oligonucleotide. 30.The oligonucleotide of claim 29, wherein the internucleotide linkageassociated with the seventh, eighth, ninth, tenth, eleventh, and twelfthnucleotide from the 5′ end of the oligonucleotide is phosphorothioatemodified.
 31. The oligonucleotide of any one of claims 24-30, whereineach nucleotide has either a 2′O methyl modification or phosphorothioateinternucleotide linkage.
 32. The oligonucleotide of claims 24, whereinonly one nucleotide has both a 2′O methyl modification and aphosphorothioate internucleotide linkage.
 33. The oligonucleotide ofclaim 24, wherein only one nucleotide has a 2′-modified nucleotide. 34.The oligonucleotide of claim 33, wherein the 2′-modification is selectedfrom the group of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and2′-O-N-methylacetamido (2′-O-NMA).
 35. A stable self-assemblingnanostructure, comprising an antisense oligonucleotide of 18-19nucleotides in length comprising TGGGAGTAGATGAGGTAC (SEQ ID NO. 4),wherein the antisense oligonucleotide is associated with a core.
 36. Thenanostructure of claim 35, wherein the antisense oligonucleotide is 18nucleotides in length.
 37. The nanostructure of claim 35 or 36, whereinthe antisense oligonucleotide has phosphodiester internucleotidelinkages.
 38. The nanostructure of claim 37, wherein less than all ofthe internucleotide linkages are phosphodiester.
 39. The nanostructureof claim 35 or 36, wherein the antisense oligonucleotide hasphosphorothioate internucleotide linkages.
 40. The nanostructure ofclaim 39, wherein less than all of the internucleotide linkages arephosphorothioate.
 41. The nanostructure of any one of claims 35-40,wherein the antisense oligonucleotide has 2′O methyl modifications. 42.The nanostructure of claim 41, wherein less than all of the nucleotidesinclude a 2′O methyl modification.
 43. The nanostructure of claim 36,wherein the antisense oligonucleotide has 17 internucleotide linkagesand wherein the 6 central internucleotide linkages are phosphorothioate.44. The nanostructure of claim 43, wherein the first 6 internucleotidelinkages at the 5′ end of the oligonucleotide are phosphodiesterinternucleotide linkages.
 45. The nanostructure of claim 44, wherein thefirst 6 nucleotides at the 5′ end of the oligonucleotide are 2′O methylmodified nucleotides.
 46. The nanostructure of claim 44 or 45, whereinthe last 5 nucleotides at the 3′ end of the oligonucleotide are 2′Omethyl modified nucleotides.
 47. The nanostructure of claim 36, whereinthe antisense oligonucleotide is selected from the group consisting ofT-G-G-G-A-G-T-A-G-A-T-G-A-G-G-T-A-C (SEQ ID NO. 4),mUmGmGmGmAmGmUmAmGmAmUmGmAmGmGmUmAmC (SEQ ID NO. 10, Oligo 3742),T*G*G*G*A*G*T*A*G*A*T*G*A*G*G*T*A*C (SEQ ID NO. 9, Oligo 3500),mUmGmGmGmAmGT*A*G*A*T*G*mAmGmGmUmAmC (SEQ ID NO. 16, Oligo 3534), andmU*mG*mG*mG*mA*mG*T*A*G*A*T*G*mA*mG*mG*mU*mA*mC (SEQ ID NO. 18, Oligo3509) wherein—refers to a phosphodiester bond, * refers to aphosphorothioate bond, and m refers to a O methyl.
 48. The nanostructureof any one of claims 35-47, wherein the antisense oligonucleotide islinked to the exterior of the core.
 49. The nanostructure of any one ofclaims 35-47, wherein the nanostructure includes 2-1,000 copies of theantisense oligonucleotide.
 50. The nanostructure of any one of claims35-48, wherein the nanostructure includes at least one oligonucleotidestructurally distinct from the antisense oligonucleotide.
 51. Thenanostructure of any one of claims 35-50, wherein the antisenseoligonucleotide has its 5′-terminus exposed to the outside surface ofthe nanostructure.
 52. The nanostructure of any one of claims 35-50,wherein the antisense oligonucleotide has its 3′-terminus exposed to theoutside surface of the nanostructure.
 53. The nanostructure of any oneof claims 35-50, wherein the antisense oligonucleotide is positionedlaterally on the surface of the nanostructure.
 54. The nanostructure ofany one of claims 35-53, wherein the antisense oligonucleotide isindirectly linked to the core through a linker.
 55. The nanostructure ofany one of claims 35-54, wherein the antisense oligonucleotide isindirectly linked to the core through more than one linker.
 56. Thenanostructure of any one of claims 35-55, wherein the core is a solid orhollow core.
 57. The nanostructure of any one of claims 35-55, whereinthe core is inert, paramagnetic or superparamagnetic.
 58. Thenanostructure of any one of claims 35-55, wherein the core is a solidcore.
 59. The nanostructure of claim 58, wherein the solid core iscomprised of noble metals, including gold and silver, transition metalsincluding iron and cobalt, metal oxides including silica, polymers orcombinations thereof.
 60. The nanostructure of claim 58, wherein thecore is a polymeric core and wherein the polymeric core is comprised ofamphiphilic block copolymers, hydrophobic polymers includingpolystyrene, poly(lactic acid), poly(lactic co-glycolic acid),poly(glycolic acid), poly(caprolactone) and other biocompatiblepolymers.
 61. The nanostructure of any one of claims 35-55, wherein thecore is a liposomal core.
 62. A composition comprising the compound ofany one of claims 1-12, the oligonucleotide of any one of claims 13-34or the nanostructure of any one of claims 35-61, further comprising atherapeutic agent for treating a TNF disorder associated with thenanostructure.
 63. The composition of claim 62, wherein the therapeuticagent is linked to the oligonucleotide.
 64. A method for treating a TNFdisorder, comprising: administering to a subject having a TNF disorder acomposition comprising the compound of any one of claims 1-12, theoligonucleotide of any one of claims 13-34 or the nanostructure of anyone of claims 35-61 in an effective amount to treat the TNF disorder.65. The method of claim 64, wherein the TNF disorder is selected fromthe group consisting of an autoimmune disease, an infectious disease,transplant rejection or graft-versus-host disease, malignancy, apulmonary disorder, an intestinal disorder, a cardiac disorder, sepsis,a spondyloarthropathy, a metabolic disorder, anemia, pain, a hepaticdisorder, a skin disorder, a nail disorder, rheumatoid arthritis,psoriasis, psoriasis in combination with psoriatic arthritis, ulcerativecolitis, Crohn's disease, vasculitis, Behcet's disease, ankylosingspondylitis, asthma, chronic obstructive pulmonary disorder (COPD),idiopathic pulmonary fibrosis (IPF), restenosis, diabetes, anemia, pain,a Crohn's disease-related disorder, juvenile rheumatoid arthritis (JRA),a hepatitis C virus infection, psoriatic arthritis, and chronic plaquepsoriasis.
 66. The method of claim 65, wherein the autoimmune disorderis selected from the group consisting of rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, gouty arthritis, allergy,multiple sclerosis, autoimmune diabetes, autoimmune uveitis, andnephritic syndrome.
 67. A method for reducing TNF levels in vivo,comprising: administering to a subject a composition comprising thecompound of any one of claims 1-12, the oligonucleotide of any one ofclaims 13-34 or the nanostructure of any one of claims 35-61 in aneffective amount to reduce TNF levels in vivo.
 68. A stableself-assembling nanostructure, comprising an antisense oligonucleotideof 18-19 nucleotides in length comprising TGGGAGTAGATGAGGTAC (SEQ ID NO.4), wherein a hydrophobic group at the 3′ or 5′ terminus self-associatesto form the core of the nanostructure in water or other suitablesolvents.
 69. The stable self-assembling nanostructure of claim 68,wherein the oligonucleotide is at concentrations above 5 μM in DNase andRNase free water or other suitable solvents.
 70. The stableself-assembling nanostructure of claim 68, wherein the antisenseoligonucleotide is 18 nucleotides in length.
 71. The stableself-assembling nanostructure of claim 68, wherein the antisenseoligonucleotide has phosphodiester internucleotide linkages.
 72. Thestable self-assembling nanostructure of claim 68, wherein less than allof the internucleotide linkages are phosphodiester.